Methods and compositions to reduce or eliminate transmission of a transgene

ABSTRACT

Genetic constructs and methods are disclosed for the production, maintenance and control of transgenes in transgenic eukaryotic organisms that undergo meiosis in which pollen or sperm can be outcrossed; this includes: transgenic animals, plant cells, plant tissues and whole plants. More specifically, this invention relates to the control of transgene transmission by male and/or female gametes or gametophytes using a gametophytic sterility trait (GST). The genetic constructs and methodologies of the present invention provide the ability to control the undesired spread of transgenes. In addition, this invention also provides the tools and methodologies to enrich a plant or other eukaryotic genome for dispersed and/or stable transposition events.

CROSS REFERENCES TO RELATED APPLCATIONS

[0001] This application claims priority to Provisional Application60/185,524, filed Feb. 28, 2000, which is hereby incorporated byreference in its entirety.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

[0002] This invention was partially made with government support underGrant Nos. NIH GM R01 GM38148 and NSF 61-2558.

FIELD OF THE INVENTION

[0003] This invention relates generally to the production, maintenanceand control of transgenes in transgenic eukaryotic organsims thatundergo meiosis in which pollen or sperm can be outcrossed; thisincludes: transgenic animals, plant cells, plant tissues and wholeplants. More specifically, this invention relates to the control oftransgene transmission by male and/or female gametes or gametophytes.The genetic constructs and methodologies of the present inventionprovide the ability to control the undesired spread of transgenes. Inaddition, this invention also provides the tools and methodologies toenrich a plant genome, or any other eukaryotic genome, for dispersedand/or stable transposition events.

BACKGROUND OF THE INVENTION

[0004] All publications and patent applications herein are incorporatedby reference to the same extent as if each individual publication orpatent application was specifically and individually indicated to beincorporated by reference.

[0005] Transgenic crops and the application of biotechnology aredramatically altering seed and agrochemical businesses throughout theworld. The seeds of commercially important crops have been geneticallyengineered to be resistant to herbicides and pests, especially insectpests. According to surveys by the United States Department ofAgriculture (June, 2000), genetically modified corn, soybeans and cottonwere grown on approximately 25%, 54% and 61%, respectively, of the totalU.S. acres for each crop in 2000.

[0006] The uncontrolled transmission of heterologous traits incommercially important crop plants is currently a major concernthroughout the world and especially within the agricultural community.The undesired dissemination of transgenic pollen may unintentionallyharm beneficial insects and may result in the spread of transgenes torelated plant species leading to the contamination of food products andthe production of herbicide- and pesticide-resistant weedy species.

[0007] The biotechnology industry is interested in transferring traitssuch as tolerances to drought, insects, diseases, salinity, frost andherbicides into cultivated plants which might confer an adaptiveadvantage over wild plants. Several crop species are known to becross-compatible with wild species and it is possible that these traitscould be inadvertently transferred to wild weedy relatives throughsexual hybridization leading to possible economic and ecological harm.Since most forage and turf grasses have undergone relatively littledomestication and may even be considered weeds in certain instances(e.g., bermudagrass), there is a high probability of greater problems inthe ultimate release and use of such genetically transformed plants.Because forage grasses, in general, are not highly domesticated, possessweedy characteristics, and are highly outcrossing, special difficultiesmay be encountered in the ultimate release of transgenic forage grasses.

[0008] It would be highly desirable to have a method to prevent theundesired transmission of heterologous traits in commercially importantcrop plants. If this could be achieved, genetic leakage of heterologoustraits would be brought under control and the spread of these traits toundesired recipients would be curtailed. Thus, the need exits for agenetic system that selects against male or female gametophytescontaining transgenes, thereby preventing, eliminating or reducing theundesired transmission of heterologous traits. In particular, there is aneed for a genetic system which allows for the transmission ofnon-transgenic (i.e., wild type) gametophytes while preventing thetransmission of the transgenic (i.e., heterologous) gametophytes fromthe same plant.

[0009] Thus, an object of this invention is to provide recombinantnucleic acid constructs and methods for controlling, reducing oreliminating the undesired transmission of heterologous traits incommercially important crop plants.

SUMMARY OF THE INVENTION

[0010] The instant invention is directed to genetic constructs andmethods for controlling the spread of heterologous traits in plants.Control is achieved by providing a sex-specific promoter operably linkedto a suicide gene that selects against male or female gametes containingthe suicide gene. The suicide gene locus is termed the “gametophyticsuicide trait” (GST) (FIG. 1A). By linking a transgene of interest to asuicide gene under the control of a sex-specific promoter, transmissionof the transgene to progeny is effectively eliminated, reduced orprevented because no gametes bearing the GST will be produced.

[0011] In one aspect, the invention can be said to broadly consist of asuicide gene under the control of a pollen-specific promoter linked to atransgene of interest. The transgene complex can be introduced into avirgin plant genome and plants can be selected which are hemizygous forthe transgene complex. The only pollen produced by the hemizygous plantwill lack the transgene complex due to its physical linkage to thesuicide gene. Uncontrolled spread of the heterologous trait encoded bythe transgene complex is thereby prevented because no pollen containingthe transgene complex is produced.

[0012] A second aspect of the invention is based on placing the GST inclose proximity to a transposon to produce selective enrichment ofdispersed transposition events in progeny cells since only those gameteslacking the GST locus will be viable. Since a fraction of the progenycells produced from viable gametes will have undergone transpositionevents, selective enrichment of dispersed transposition events isachieved because the transposon is necessarily no longer linked to theGST (the GST destroys those gametes that inherit the GST gene locus)(FIG. 1B).

[0013] Thus, the present invention provides genetic systems which can beused for the elimination of a GST transgene complex and for theselection for unlinked transpositions. By using the GST together withany transgene, one can completely eliminate male (or female in the caseof a female gametophytic-specific promoter:suicide construct)transmission of both the GST and the associated transgene (FIG. 2).

[0014] This invention provides nucleic acid constructs comprising a malegamete- or female gamete-specific promoter operably linked to a suicidegene, wherein said promoter and said suicide gene combination is linkedto a gene of interest.

[0015] This invention provides nucleic acid constructs comprising a malegamete- or female gamete-specific promoter operably linked to a suicidegene, wherein the promoter and the suicide gene combination is linked toa gene of interest. This invention further provides such nucleic acidconstructs wherein the promoter is selected from the group consisting ofa pollen-specific promoter and an ovule-specific promoter. Thisinvention still further provides such nucleic acid constructs whereinthe suicide gene is selected from the group consisting of bamase,tasselseed2 and diphtheria toxin A gene. This invention also providessuch nucleic acid constructs wherein the gene of interest is selectedfrom the group consisting of a nucleic acid encoding herbicideresistance, antibiotic resistance, insecticide resistance, nitrogenfixation, improved nutrition and cellulose content.

[0016] This invention provides nucleic acid constructs comprising apollen-specific promoter or an ovule-specific promoter operably linkedto a suicide gene selected from the group consisting of barnase,tasselseed2 and diphtheria toxin A gene; wherein the promoter and thesuicide gene combination is linked to a gene of interest selected fromthe group consisting of a gene coding for herbicide resistance,antibiotic resistance, insecticide resistance, nitrogen fixation,improved nutrition and cellulose content or other agronomic trait ofinterest.

[0017] This invention provides methods for reducing or eliminating maletransmission of a transgene locus in a plant comprising:

[0018] a) transforming a plant cell with a nucleic acid construct inwhich a male gamete-specific promoter is operably linked to a suicidegene, wherein said promoter and said suicide gene combination is linkedto a heterologous polynucleotide;

[0019] b) propagating said transformed plant cell through meiosis toproduce male gametes lacking said transgene locus.

[0020] This invention also provides methods for reducing or eliminatingmale transmission of a transgene locus in a plant comprising:

[0021] a) transforming a plant cell with a nucleic acid construct inwhich a pollen-specific promoter is operably linked to a suicide gene;

[0022] i) wherein said suicide gene is selected from the groupconsisting of bamase, tasselseed2 and diphtheria toxin A gene;

[0023] ii) wherein said promoter and said suicide gene combination islinked to a heterologous polynucleotide;

[0024] iii) wherein said heterologous polynucleotide is selected fromthe group consisting of DNA encoding herbicide resistance, antibioticresistance, insecticide resistance, nitrogen fixation, improvednutrition and cellulose content;

[0025] b) propagating said transformed plant cell through meiosis toproduce male gametes lacking said transgene locus.

[0026] This invention also provides methods for reducing or eliminatingfemale transmission of a transgene locus in a plant comprising:

[0027] a) transforming a plant cell with a nucleic acid construct inwhich a female gamete-specific promoter is operably linked to a suicidegene, wherein said promoter and said suicide gene combination is linkedto a heterologous polynucleotide;

[0028] b) propagating said transformed plant cell through meiosis toproduce female gametes lacking said transgene locus.

[0029] This invention further provides methods for reducing oreliminating female transmission of a transgene locus in a plantcomprising:

[0030] a) transforming a plant cell with a nucleic acid construct inwhich an ovule-specific promoter is operably linked to a suicide gene;

[0031] i) wherein said suicide gene is selected from the groupconsisting of barnase, tasselseed2 and diphtheria toxin A gene;

[0032] ii) wherein said promoter and said suicide gene combination islinked to a heterologous polynucleotide;

[0033] iii) wherein said heterologous polynucleotide is selected fromthe group consisting of DNA encoding herbicide resistance, antibioticresistance, insecticide resistance, nitrogen fixation, improvednutrition and cellulose content.

[0034] b) propagating said transformed plant cell through meiosis toproduce female gametes lacking said transgene locus.

[0035] This invention also provides transformed plant cells produced bythe methods of the present invention wherein the transformed plant cellsare hemizygotic for the nucleic acid construct.

[0036] This invention provides nucleic acid constructs comprising a malegamete- or female gamete-specific promoter operably linked to a suicidegene wherein said promoter and said suicide gene combination is linkedto a transposable element. This invention also provides such nucleicacid constructs which further comprise one or more transposase genes.This invention further provides such nucleic acid constructs whichfurther comprise one or more genes of interest. This invention stillfurther provides such nucleic acid constructs wherein the gene ofinterest is associated with the transposable element.

[0037] This invention provides nucleic acid constructs in which apollen-specific promoter or an ovule-specific promoter is operablylinked to a suicide gene selected from the group consisting of bamase,tasselseed2 and diphtheria toxin A gene; wherein said promoter and saidsuicide gene combination is linked to a transposon, wherein saidtransposon comprises a selectable marker selected from the groupconsisting of a gene coding for herbicide resistance, antibioticresistance, insecticide resistance, nitrogen fixation, improvednutrition and cellulose content. This invention also provides suchnucleic acid constructs wherein the promoter is selected from the groupconsisting of a pollen-specific promoter and an ovule-specific promoter.This invention also provides such nucleic acid constructs wherein thesuicide gene is selected from the group consisting of barnase,tasselseed2 and diphtheria toxin A gene.

[0038] The present invention provides methods for enriching dispersedtransposition events in a population of plant cell progeny comprising:

[0039] a) transforming a plant cell with the nucleic acid construct ofany one of the aforementioned nucleic acid constructs to produce atransformed plant cell;

[0040] b) propagating said transformed plant cell through meiosis toproduce plant cell progeny in which dispersed transposition events areenriched.

[0041] The present invention also provides such methods which includethe additional step of isolating the plant cell progeny in whichdispersed transposition events are enriched. The present invention alsoprovides plant cells and plants which contain dispersed transpositionevents and, particularly, the plant cells and plants are hemizygotic forthe nucleic acid.

[0042] The present invention provides nucleic acid constructs comprisinga first promoter wherein the first promoter is a male gamete- or femalegamete-specific promoter operably linked to a suicide gene and furthercomprising a nucleic acid encoding a transposase and a nucleic acidencoding a transposon. The present invention also provides such nucleicacid constructs wherein the transposon comprises a second promoteroperably linked to a selectable marker, wherein the selectable marker isnot a suicide gene.

[0043] The present invention provides nucleic acid constructs in which apollen-specific promoter or an ovule-specific promoter is operablylinked to a suicide gene selected from the group consisting of barnase,tasselseed2 and diphtheria toxin A gene, wherein said promoter and saidsuicide gene combination is linked to a nucleic acid encodingtransposase; wherein said promoter and said suicide gene combinationlinked to said nucleic acid encoding transposase comprise a transgenelocus which further comprises a transposon; wherein said transposoncomprises a polynucleotide sequence encoding a member selected from thegroup consisting of herbicide resistance, antibiotic resistance,insecticide resistance, nitrogen fixation, improved nutrition andcellulose content. The present invention provides such nucleic acidconstructs wherein the promoter is selected from the group consisting ofa pollen-specific promoter and an ovule-specific promoter. The presentinvention further provides such nucleic acid constructs wherein thesuicide gene is selected from the group consisting of bamase,tasselseed2 and diphtheria toxin A gene. The present invention alsoprovides such nucleic acid constructs wherein the transposon comprises apolynucleotide sequence encoding a member selected from the groupconsisting of herbicide resistance, antibiotic resistance, insecticideresistance, nitrogen fixation, improved nutrition and cellulose content.

[0044] The present invention also provides methods for enriching stablydispersed transposition events in a population of plant cell progenycomprising:

[0045] a) transforming a plant cell with a nucleic acid constructs ofthe present invention to produce a transformed plant cell;

[0046] b) propagating said transformed plant cell through meiosis toproduce plant cell progeny in which stably dispersed transpositionevents are enriched.

[0047] The present invention also provide such methods furthercomprising the step of isolating the plant cell progeny in which thestably dispersed transposition events are enriched. The inventionfurther provides plant cells isolated by such methods and plantsproduced from the plant cells.

[0048] This invention provides vectors comprising the nucleic acidconstructs of the present invention as well as host cells, recombinantplant cells and transgenic plants comprising the vectors of the presentinvention. More particularly, this invention provides such cells andtransgenic plants which are hemizygotic for the nucleic acid constructs.

[0049] Other objects, advantages and features of the present inventionbecome apparent to one skilled in the art upon reviewing thespecification and the drawings provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The foregoing summary, as well as the following detaileddescription of the invention, will be better understood when read inconjunction with the appended drawings. For the purpose of illustratingthe invention, there are shown in the drawings embodiment(s) which arepresently preferred. It should be understood, however, that theinvention is not limited to the precise arrangements andinstrumentalities shown.

[0051] In the drawings:

[0052]FIG. 1A shows an illustrative GST (Gametophytic Sterility Trait)Construct in which a pollen-specific promoter is operably linked to asuicide gene. The GST construct can be physically linked to a gene ofinterest to form a transgene complex. The GST construct is used toprevent or eliminate transmission of the gene of interest.

[0053]FIG. 1B shows an illustrative GST construct linked to atransposable element and a transposase source. This construct can beused to enrich a population of plant cell progeny for stably dispersedtransposons.

[0054]FIG. 2 shows a generalized strategy for eliminating a transgenecomplex from meiotic products and to select for dispersedtranspositions.

[0055]FIGS. 3A and 3B show schematics of GST constructs.

[0056]FIGS. 4A and 4B show schematics of pYU904 and pYU905 constructs,respectively.

[0057]FIG. 5 shows a schematic of pYU846 transposase construct.

DETAILED DESCRIPTION

[0058] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods and materials are described.

[0059] It will be appreciated from the above that the tools and methodsof the present invention have application to all plants that producegametes. Such plants include, but are not limited to, forage grasses,turf grasses, forage legumes, vegetables, field crops, trees andornamental flowers.

[0060] Definitions

[0061] As used herein, the term “allele” refers to any of severalalternative forms of a gene.

[0062] As used herein, the term “crop plant” refers to any plant grownfor any commercial purpose, including, but not limited to the followingpurposes: seed production, hay production, ornamental use, fruitproduction, berry production, vegetable production, oil production,protein production, forage production, animal grazing, golf courses,lawns, flower production, landscaping, erosion control, green manure,improving soil tilth/health, producing pharmaceutical products/drugs,producing food additives, smoking products, pulp production and woodproduction.

[0063] As used herein, the term “cross pollination” or “cross-breeding”refer to the process by which the pollen of one flower on one plant isapplied (artificially or naturally) to the ovule (stigma) of a flower onanother plant.

[0064] As used herein, the term “cultivar” refers to a variety, strainor race of plant that has been produced by horticultural or agronomictechniques and is not normally found in wild populations.

[0065] The term “dispersed transposition event” refers to the movementof a transposon such that it is no longer linked (i.e. in closeproximity) to the transposon launch site (donor site).

[0066] The term “female” refers to a plant that produces ovules. Femaleplants generally produce seeds after fertilization. A plant designatedas a “female plant” may contain both male and female sexual organs.Alternatively, the “female plant” may only contain female sexual organseither naturally (e.g., in dioecious species) or due to emasculation(e.g., by detasselling).

[0067] As used herein, the term “filial generation” refers to any of thegenerations of cells, tissues or organisms following a particularparental generation. The generation resulting from a mating of theparents is the first filial generation (designated as “F1” or “F1”),while that resulting from crossing of F1 individuals is the secondfilial generation (designated as “F2” or “F2”).

[0068] The term “gamete” refers to a reproductive cell whose nucleus(and often cytoplasm) fuses with that of another gamete of similarorigin but of opposite sex to form a zygote, which has the potential todevelop into a new individual. Gametes are haploid and aredifferentiated into male and female.

[0069] The term “gene” refers to any segment of DNA associated with abiological function. Thus, genes include, but are not limited to, codingsequences and/or the regulatory sequences required for their expression.Genes can also include nonexpressed DNA segments that, for example, formrecognition sequences for other proteins. Genes can be obtained from avariety of sources, including cloning from a source of interest orsynthesizing from known or predicted sequence information, and mayinclude sequences designed to have desired parameters.

[0070] As used herein, the term “genotype” refers to the genetic makeupof an individual cell, cell culture, tissue, plant, or group of plants.

[0071] As used herein, the term “hemizygous” refers to a cell, tissue ororganism in which a gene is present only once in a genotype, as a genein a haploid cell or organism, a sex-linked gene in the heterogameticsex, or a gene in a segment of chromosome in a diploid cell or organismwhere its partner segment has been deleted.

[0072] A “heterologous polynucleotide” or a “heterologous nucleic acid”or an “exogenous DNA segment” refers to a polynucleotide, nucleic acidor DNA segment that originates from a source foreign to the particularhost cell, or, if from the same source, is modified from its originalform. Thus, a heterologous gene in a host cell includes a gene that isendogenous to the particular host cell, but has been modified. Thus, theterms refer to a DNA segment which is foreign or heterologous to thecell, or homologous to the cell but in a position within the host cellnucleic acid in which the element is not ordinarily found. Exogenous DNAsegments are expressed to yield exogenous polypeptides.

[0073] A “heterologous trait” refers to a phenotype imparted to atransformed host cell or transgenic organism by an exogenous DNAsegment, heterologous polynucleotide or heterologous nucleic acid.

[0074] As used herein, the term “heterozygote” refers to a diploid orpolyploid individual cell or plant having different alleles (forms of agiven gene) present at least at one locus.

[0075] As used herein, the term “heterozygous” refers to the presence ofdifferent alleles (forms of a given gene) at a particular gene locus.

[0076] As used herein, the term “homozygote” refers to an individualcell or plant having the same alleles at one or more loci.

[0077] As used herein, the term “homozygous” refers to the presence ofidentical alleles at one or more loci in homologous chromosomalsegments.

[0078] As used herein, the term “hybrid” refers to any individual cell,tissue or plant resulting from a cross between parents that differ inone or more genes.

[0079] As used herein, the term “inbred” or “inbred line” refers to arelatively true-breeding strain.

[0080] As used herein, the term “line” is used broadly to include, butis not limited to, a group of plants vegetatively propagated from asingle parent plant, via tissue culture techniques or a group of inbredplants which are genetically very similar due to descent from a commonparent(s). A plant is said to “belong” to a particular line if it (a) isa primary transformant (T0) plant regenerated from material of thatline; (b) has a pedigree comprised of a T0 plant of that line; or (c) isgenetically very similar due to common ancestry (e.g., via inbreeding orselfing). In this context, the term “pedigree” denotes the lineage of aplant, e.g. in terms of the sexual crosses effected such that a gene ora combination of genes, in heterozygous (hemizygous) or homozygouscondition, imparts a desired trait to the plant.

[0081] As used herein, the term “locus” (plural: “loci”) refers to anysite that has been defined genetically. A locus may be a gene, or partof a gene, or a DNA sequence that has some regulatory role, and may beoccupied by different sequences.

[0082] The term “male” refers to a plant that produces pollen grains.The “male plant” generally refers to the sex that produces gametes forfertilizing ova. A plant designated as a “male plant” may contain bothmale and female sexual organs. Alternatively, the “male plant” may onlycontain male sexual organs either naturally (e.g., in dioecious species)or due to emasculation (e.g., by removing the ovary).

[0083] As used herein, the term “mass selection” refers to a form ofselection in which individual plants are selected and the nextgeneration propagated from the aggregate of their seeds.

[0084] As used herein, the terms “nucleic acid” or “polynucleotide”refer to deoxyribonucleotides or ribonucleotides and polymers thereof ineither single- or double-stranded form. Unless specifically limited, theterms encompass nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g. degenerate codon substitutions) and complementarysequences as well as the sequence explicitly indicated. Specifically,degenerate codon substitutions may be achieved by generating sequencesin which the third position of one or more selected (or all) codons issubstituted with mixed-base and/or deoxyinosine residues (Batzer et al.(1991) Nucleic Acid Res. 19:5081; Ohtsuka et al. (1985) J. Biol. Chem.260:2605-2608; Cassol et al. (1992); Rossolini et al. (1994) Mol. Cell.Probes 8:91-98). The term nucleic acid is used interchangeably withgene, cDNA, and mRNA encoded by a gene.

[0085] As used herein, a DNA segment is referred to as “operably linked”when it is placed into a functional relationship with another DNAsegment. For example, DNA for a signal sequence is operably linked toDNA encoding a polypeptide if it is expressed as a preprotein thatparticipates in the secretion of the polypeptide; a promoter or enhanceris operably linked to a coding sequence if it stimulates thetranscription of the sequence. Generally, DNA sequences that areoperably linked are contiguous, and in the case of a signal sequenceboth contiguous and in reading phase. However, enhancers need not becontiguous with the coding sequences whose transcription they control.Linking is accomplished by ligation at convenient restriction sites orat adapters or linkers inserted in lieu thereof.

[0086] As used herein, the term “open pollination” refers to a plantpopulation that is freely exposed to some gene flow, as opposed to aclosed one in which there is an effective barrier to gene flow.

[0087] As used herein, the terms “open-pollinated population” or“open-pollinated variety” refer to plants normally capable of at leastsome cross-fertilization, selected to a standard, that may showvariation but that also have one or more genotypic or phenotypiccharacteristics by which the population or the variety can bedifferentiated from others. A hybrid, which has no barriers tocross-pollination, is an open-pollinated population or anopen-pollinated variety.

[0088] As used herein, the term “ovule” refers to the femalegametophyte, whereas the term “pollen” means the male gametophyte.

[0089] As used herein, the term “ovule-specific promoter” refers broadlyto a nucleic acid sequence that regulates the expression of nucleic acidsequences selectively in the cells or tissues of a plant essential toovule formation and/or function and/or limits the expression of anucleic acid sequence to the period of ovule formation in a plant.

[0090] As used herein, the term “pollen-specific promoter” refersbroadly to a nucleic acid sequence that regulates the expression ofnucleic acid sequences selectively in the cells or tissues of a plantessential to pollen formation and/or function and/or limits theexpression of a nucleic acid sequence to the period of pollen formationin the plant.

[0091] As used herein, the term “phenotype” refers to the observablecharacters of an individual cell, cell culture, plant, or group ofplants which results from the interaction between that individual'sgenetic makeup (i.e., genotype) and the environment.

[0092] As used herein, the term “plant” refers to whole plants, plantorgans (e.g., leaves, stems, roots, etc.), seeds and plant cells andprogeny of it. The class of plants that can be used in the methods ofthe invention is generally as broad as the class of higher plantsamenable to transformation techniques, including both monocotyledonousand dicotyledonous plants.

[0093] As used herein, the term “promoter” refers to a region of DNAinvolved in binding RNA polymerase to initiate transcription.

[0094] As used herein, the term “recombinant” refers to a cell, tissueor organism that has undergone transformation with recombinant DNA. Theoriginal recombinant is designated as “R0” or “R₀.” Selfing the R0produces a first transformed generation designated as “R1” or “R₁.”

[0095] As used herein, the term “self-incompatible” means the failure,following mating or pollination, of a male gamete and a female gamete toachieve fertilization, where each of them is capable of uniting withother gametes of the breeding group after similar mating or pollination(Mather, J. Genet. 25:215-235 (1943)).

[0096] As used herein, the term “self pollinated” or “self-pollination”means the pollen of one flower on one plant is applied (artificially ornaturally) to the ovule (stigma) of the same or a different flower onthe same plant.

[0097] As used herein, the term “stably dispersed transposition event”refers to a dispersed transposition that does not undergo furthertranspositions such as secondary transposition events.

[0098] As used herein, the term “suicide gene” refers to any gene thatexpresses a product that is fatal to the cell expressing the suicidegene.

[0099] As used herein, the term “synthetic” refers to a set of progeniesderived by intercrossing a specific set of clones or seed-propagatedlines. A synthetic may contain mixtures of seed resulting from cross-,self-, and sib-fertilization.

[0100] As used herein, the term “transformation” refers to the transferof nucleic acid (i.e., a nucleotide polymer) into a cell. Asused-herein, the term “genetic transformation” refers to the transferand incorporation of DNA, especially recombinant DNA, into a cell.

[0101] As used herein, the term “transformant” refers to a cell, tissueor organism that has undergone transformation. The original transformantis designated as “T0” or “T₀.” Selfing the T0 produces a firsttransformed generation designated as “T1” or “T₁.”

[0102] As used herein, the term “transgene” refers to a nucleic acidthat is inserted into an organism, host cell or vector in a manner thatensures its function.

[0103] As used herein, the term “transgenic” refers to cells, cellcultures, organisms, plants, and progeny of plants which have received aforeign or modified gene by one of the various methods oftransformation, wherein the foreign or modified gene is from the same ordifferent species than the species of the plant, or organism, receivingthe foreign or modified gene.

[0104] As used herein, the term “transposase” refers to an enzyme,enzymes, or more generally, a molecule or molecules that catalyze atransposition event.

[0105] As used herein, the term “tansposition event” refers to themovement of a transposon from a donor site to a target site.

[0106] As used herein, the term “transposon” refers to a geneticelement, including but not limited to segments of DNA or RNA that canmove from one chromosomal site to another.

[0107] As used herein, the term “variety” refers to a subdivision of aspecies, consisting of a group of individuals within the species thatare distinct in form or function from other similar arrays ofindividuals.

[0108] As used herein, the term “vector” refers broadly to any plasmidor virus encoding an exogenous nucleic acid. The term should also beconstrued to include non-plasmid and non-viral compounds whichfacilitate transfer of nucleic acid into virions or cells, such as, forexample, polylysine compounds and the like. The vector may be a viralvector that is suitable as a delivery vehicle for delivery of thenucleic acid, or mutant thereof, to a cell, or the vector may be anon-viral vector which is suitable for the same purpose. Examples ofviral and non-viral vectors for delivery of DNA to cells and tissues arewell known in the art and are described, for example, in Ma et al.(1997, Proc. Natl. Acad. Sci. U.S.A. 94:12744-12746). Examples of viralvectors include, but are not limited to, a recombinant vaccinia virus, arecombinant adenovirus, a recombinant retrovirus, a recombinantadeno-associated virus, a recombinant avian pox virus, and the like(Cranage et al., 1986, EMBO J. 5:3057-3063; International PatentApplication No. WO94/17810, published Aug. 18, 1994; InternationalPatent Application No. WO94/23744, published Oct. 27, 1994). Examples ofnon-viral vectors include, but are not limited to, liposomes, polyaminederivatives of DNA, and the like.

OVERVIEW OF THE INVENTION

[0109] Constructs and methods are described for the destruction of themale or female gametophyte (microspores or megaspores) for the purposesof eliminating transmission of a transgene locus (gene of interest). Inone embodiment, microspore destruction is genetically engineered using apollen-specific promoter fused to an appropriate suicide gene or throughthe use of genetic mutations that are unable to be transmitted throughone of the sexes.

[0110] When hemizygous, eliminating transmission of a transgene locus isachieved by linking a gene of interest to a suicide gene under thecontrol of a male or female-specific promoter. This construct, termedthe “gametophytic suicide trait” (GST) induces cell death that isrestricted to microspores or megaspores, thereby effectively reducing oreliminating transmission of the gene of interest that is linked to theGST. Since the GST/transgene construct is hemizygous, 50% of the pollengrains will be viable and non-transgenic. Thus, the transmission of atransgene can be controlled while permitting pollination to occur so asto achieve fertilization and ultimately obtain a seed supply forplanting or food use. Since many plants produce an over-abundance ofpollen, the loss of 50% of the pollen produced will not adversely affectseed set for most plant species. As one example, corn (Zea mays)produces as many as 10⁷ pollen grains/day for a plant in the peak of a 7day flowering period (Coe et al., The Genetics of Corn, In: Corn andCorn Improvement. Third Edition. Editors: Sprague et al., (1988) pp.81-258).

[0111] Eliminating male transmission of a transgene locus can also beused as a novel strategy to enrich for dispersed and/or stabletransposition events. This is accomplished by engineering a “transgenecomplex” containing a transposable element and/or transposase gene alongwith a “gametophytic suicide trait” (GST). The GST induces cell deaththat is restricted to microspores, severely reducing male transmissionof nearby chromosomal regions and other transgenes, including thetransposon (donor element) and/or transposase gene within the transgenecomplex.

[0112] When the transgene complex is heterozygous in the male parent,approximately 50% of the microspores will undergo destruction, therebypreventing male transmission of the transgene complex and greatlyreducing male transmission of linked transposed elements. Thiselimination of the transposon donor site and nearby transpositions hasthe net effect of enriching for pollen containing unlinkedtranspositions and transposed elements that have recombined with thedonor transgene complex.

[0113] Surviving transposition events can be readily selected inoffspring by including an herbicide selectable marker gene within thetransposable element. The GST can also be physically linked to thetransposase source to eliminate gametes containing a source oftransposase. This arrangement prevents transmission of the transposasesource to gametes, thereby stabilizing insertions in subsequentgenerations.

[0114] All three components—the suicide gene or mutation, thetransposase source and the transposon—can be engineered as a unit toprovide a robust method of generating dispersed, stable transpositions.An alternative strategy to microspore destruction is to engineer ovulesemi-sterility using a megaspore suicide trait to eliminate femaletransmission of the transgene complex.

[0115] I. Nucleic Acids

[0116] A. Promoters

[0117] There are many excellent examples of suitable promoters to drivepollen-specific expression in plants. Pollen-specific promoters havebeen identified in many plant species such as maize, rice, tomato,tobacco, Arabidopsis, Brassica, and others (Odell, T. O., et al. (1985)Nature 313:810-812; Marrs, K. A., et al, (1993) Dev Genet, Vol.14/1:27-41; Kim, (1992) Transgenic Res, Vol. 1/4:188-94; Carpenter, J.L., et al. (1992) Plant Cell Vol. 4/5:557-71; Albani, D. et al., (1992)Plant J. 2/3:331-42; Rommens, C. M., et al. (1992), Mol. Gen. Genet.,Vol. 231/3:433-41; Kloeckener-Gruissem, et al., (1992) Embo J, Vol.11/1:157-66; Hamilton, D. A. et al., (1992), Plant Mol Biol, Vol.18/2:211-18; Kyozuka, J., et al. (1991), Mol. Gen. Genet., Vol.228/1-2:40-8; Albani, D. et. al (1991) Plant Mol Biol Vol. 16/4:501-13;Twell, D. et al. (1991) Genes Dev. 5/3:496-507; Thorsness, M. K. et al.,(1991) Dev. Biol Vol. 143/1:173-84; McCormick, S. et al. (1991) Symp SocExp Biol Vol. 45:229-44; Guerrero, F. D. et al. (1990) Mol Gen Genet Vol224/2:161-8; Twell, D. et al., (1990) Development Vol. 109/3:705-13;Bichler, J. et al. (1990), Eur J Biochem Vol. 190/2:415-26; van Tunen,et al. (1990), Plant Cell Vol 2/5:393-401; Siebertz, B. et al., (1989)Plant Cell Vol 1/10:961-8; Sullivan, T. D. et al., (1989) Dev Genet Vol10/6:412-24; Chen, J. et al. (1987), Genetics Vol 116/3:469-77). Severalother examples of pollen-specific promoters can be found in GenBank.Additional promoters are also provided in U.S. Pat. Nos. 5,086,169;5,756,324; 5,633,438; 5,412,085; 5,545,546 and 6,172,279.

[0118] There are also several other eukaryotic sex-specific promoterssuitable for use in the instant invention. Examples include: the mousespermatocyte-specific Pgk-2 promoter (Ando et al. (2000) Biochem.Biophys. Res. Comm. 272/1:125-8); the PACAP testis-specific promoter(Daniel et al. (2000) Endocrinology, 141/3:1218-27); the mouse mSP-10spermatid-specific promoter (Reddi et al. (1999) Biology ofReproduction, 61/5:1256-66); the mouse sperm-specific promoter (Ramaraet al. (1998) J. Clin. Invest. 102/2:371-8); the mouse and rat Hitpromoters (vanWert et al. (1996) J. Cell. Biochem. 60/3:348-62); thehuman PRM1, PRM2 and TNP2 spermatid-specific promoters (Nelson et al.(1995) DNA Sequence 5/6:329-37); the Drosophila exu sex-specificpromoter (Crowley et al. (1995) Molec. Gen. Genet. 248/3:370-4); themouse testis ACE promoter (Zhou et al. (1995) Dev. Genet. 16/2:201-9);the rat GHRH sperrnatogenic-specific promoter (Srivastava et al. (1995)Endocrinology 136/4:1502-8); the Drosophila testis-specific promoter(Lankenau et al. (1994) Mol. Cell. Biol. 14/3:1764-75); thespermatocyte-specific hst70 gene promoter (Widlak et al. (1994) ActaBiochim. Polonica 41/2:103-5); and the mouse Prm-1 spermatid-specificpromoter (Zambrowicz et al. (1993) Proc. Nat'l. Acad. Sci. USA90/11:5071-5).

[0119] For the present invention, any promoter will be suitable if thepromoter is specific to one sex (male or female) and specifically drivesgene expression after meiosis I when homologous chromosomes haveseparated into different cells. For instance, gene expression in thetetrad stage of meiosis II, the post-mitotic division of the microsporeleading to pollen maturation, the mature pollen grain, or in thegerminating pollen grain, would be suitable for the current invention.

[0120] B. Suicide Genes

[0121] One aspect of the gametophytic suicide trait (GST) is thedirected expression of a suicide gene to kill unwanted meiotic products.Examples of genes whose expression results in cell death include, butare not limited to, those that have been described in the literatureincluding the barnase (Custers, J. B., et al., (1997) Plant Mol BiolVol. 35/6:689-99; Yazynin, S., et al., (1999) FEBS Lett Vol.452/3:351-4; Goldman, M. H, et al., (1994) Embo J Vol. 13/13:2976-84,1994), tasselseed2 (DeLong, A, et al., (1993) Cell Vol. 74/4:757-768),and the diptheria toxin A gene (Day, C. D., et al., (1995) DevelopmentVol. 121/9:2887-95). Because suicide gene expression is confined topost-meiosis I, only 50% of the gametes will be eliminated when thetransgene is hemizygous and segregates normally in meiosis. Viablegametes will not have inherited the suicide gene.

[0122] According to another aspect of the invention, semi-sterility canalso be achieved using antisense RNA technology to inhibit expression ofa gene, or genes, essential for viability of the pollen or egg. Thistechnology is discussed in more detail below.

[0123] Alternatively, mutations that are incapable of transmissionthrough one of the sexes, such as deletions that are not pollentransmitted, can also be used to achieve semi-sterility.

[0124] Specific examples of suicide genes include, but are not limitedto, the following:

[0125] Tasselseed2 (ts2. Genetic and molecular evidence shows that ts2is required for pistil elimination in both tassel and ear spikelets. ts2expression in pistil cells is coincident with loss of nuclear integrityand cell death. It is not clear how the ts2 gene product functions in acell death pathway. On the basis of its similarity to short-chainalcohol dehydrogenases, especially to hydroxysteroid dehydrogenases, twopossibilities are theorized. The ts2 product may metabolize a substrate,perhaps a steroid, required for cell viability. Alternatively, TS2action may result in the formation of a signaling molecule thatactivates a cell death response. (Calderon-Urrea et al. (1999)Development, 126:435).

[0126] Diphtheria Toxin A-chain (DTA). Diphtheria Toxin A-chain (DTA)inhibits protein synthesis, Greenfield et al., Proc. Natl. Acad.,Sci.:USA, 80:6853 (1983); Palmiter et al., Cell, 50:435 (1987).

[0127] Pectate lyase pelE. Pectate lyase pelE from Erwinia chrysanthemiEC16 degrades pectin, causing cell lysis. Keen et al., J. Bacteriology,168:595 (1986).

[0128] T-urf13 (TURF-13). T-urf13 (TURF-13) from cms-T maizemitochondrial genomes; this gene encodes a polypeptide designated URF13which disrupts mitochondrial or plasma membranes. Braun et al., PlantCell, 2:153 (1990); Dewey et al., Proc. Natl. Acad. Sci.:USA, 84:5374(1987); Dewey et al., Cell, 44:439 (1986).

[0129] Gin recombinase. Gin recombinase from phage Mu a gene encodes asite-specific DNA recombinase which will cause genome rearrangements andloss of cell viability when expressed in cells of plants. Maeser et al.,Mol. Gen. Genet., 230:170-176 (1991).

[0130] Indole acetic acid-lysine synthetase (iaaL). Indole aceticacid-lysine synthetase (iaaL) from Pseudomonas syringae encodes anenzyme that conjugates lysine to indoleacetic acid (IAA). When expressedin the cells of plants, it causes altered developments due to theremoval of IAA from the cell via conjugation. Romano et al., Genes andDevelopment, 5:438-446 (1991); Spena et al., Mol. Gen; Genet.,227:205-212 (1991); Roberto et al., Proc. Natl. Acad. Sci.:USA,87:5795-5801.

[0131] Barnase. Ribonuclease from Bacillus amyloliquefaciens, also knownas bamase, digests mRNA in those cells in which it is expressed, leadingto cell death. Mariani et al., Nature 347:737-741 (1990); Mariani etal., Nature 357:384-387 (1992).

[0132] CytA toxin gene. CytA toxin gene from Bacillus thuringiensisisraeliensis encodes a protein that is mosquitocidal and hemolytic. Whenexpressed in plant cells, it causes death of the cell due to disruptionof the cell membrane. McLean et al., J. Bacteriology, 169:1017-1023(1987); Ellar et al., U.S. Pat. No. 4,918,006 (1990).

[0133] Suitable cell death genes for use as suicide genes in othereukaryotic organisms include: human PDCD9 (programmed cell death 9) andthe Gallus gallus pro-apoptotic protein p52 (Carim et al. (1999)Cytogenetics and Cell Genetics (Switzerland) 87/1-2:85-8); the C.elegans programmed cell death genes CED-3 and EGL-1 (Hengartner et al.(1999) 54:213-22); the gene encoding the mammalian homolog of C. elegansCED-3: ICE (interleukin-1beta-converting enzyme) (Kondo et al. (1998)Investigative Ophthalmology & Visual Science 39/13:2769-74); the genesencoding ICE-like proteases Ich-1L, CPP32beta, Mch2alpha and Mch3alpha(Kondo et al. (1998) 58/5:962-7); or the mammalian cell death gene Nedd2(Kumar et al. (1997) Leukemia 11 Suppl 3:385-6).

[0134] C. Transgenes and Heterologous Nucleic Acids

[0135] Genes successfully introduced into plants using recombinant DNAmethodologies include, but are not limited to, those coding for thefollowing traits:seed storage proteins, including modified 7S legumeseed storage proteins (U.S. Pat. Nos. 5,508,468, 5,559,223 and5,576,203); herbicide tolerance or resistance (U.S. Pat. Nos. 5,498,544and 5,554,798; Powell et al., Science 232:738-743 (1986); Kaniewski etal., Bio/Tech. 8:750-754 (1990); Day et al., Proc. Natl. Acad. Sci. USA88:6721-6725 (1991)); phytase (U.S. Pat. No. 5,593,963); resistance tobacterial, fungal, nematode and insect pests, including resistance tothe lepidoptera insects conferred by the Bt gene (U.S. Pat. Nos.5,597,945 and 5,597,946; Hilder et al., Nature 330:160-163; Johnson etal., Proc. Natl. Acad. Sci. USA, 86:9871-9875 (1989); Perlak et al.,Bio/Tech. 8:939-943 (1990)); lectins (U.S. Pat. No. 5,276,269); andflower color (Meyer et al., Nature 330:677-678 (1987); Napoli et al.,Plant Cell 2:279-289 (1990); van der Krol et al., Plant Cell 2:291-299(1990)).

[0136] Of particular interest are genes that confer resistance to aherbicide. Examples include, but are not limited to, the following:

[0137] (i) An herbicide that inhibits the growing point or meristem,such as an imidazalinone or a sulfonylurea. Exemplary genes in thiscategory code for mutant ALS and AHAS enzymes as described, for example,by Lee et al., EMBO J. 7: 1241 (1988), and Miki et al., Theor. Appl.Genet. 80: 449 (1990), respectively.

[0138] (ii) Glyphosate (resistance imparted by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus phosphinothricin acetyl transferase (bar) genes), andpyridinoxy or phenoxy proprionic acids and cycloshexones (ACCaseinhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 toShah et al., which discloses the nucleotide sequence of a form of EPSPwhich can confer glyphosate resistance. A DNA molecule encoding a mutantaroA gene can be obtained under ATCC accession No. 39256, and thenucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061 to Comai. European patent application No. 0 333 033 to Kumadaet al. and U.S. Pat. No. 4,975,374 to Goodman et al. disclose nucleotidesequences of glutamine synthetase genes which confer resistance toherbicides such as L-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in Europeanapplication No. 0 242 246 to Leemans et al. De Greef et al.,Bio/Technology 7: 61 (1989), describe the production of transgenicplants that express chimeric bar genes coding for phosphinothricinacetyl transferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cycloshexones, such as sethoxydim andhaloxyfop, are the AccI-S1, Accl-S2 and Accl-S3 genes described byMarshall et al., Theor. Appl. Genet. 83: 435 (1992). The expression of aStreptomyces bar gene encoding a phosphinothricin acetyl transferase inmaize plants results in tolerance to the herbicide phosphinothricin orglufosinate (U.S. Pat. No. 5,489,520, incorporated herein by reference).

[0139] For certain target species, different antibiotic or herbicideselection markers may be preferred. Selection markers used routinely intransformation include the nptII gene which confers resistance tokanamycin and related antibiotics (Messing & Vierra, Gene 19: 259-268(1982); Bevan et al., Nature 304:184-187 (1983)), the bar gene whichconfers resistance to the herbicide phosphinothricin (White et al., NuclAcids Res 18: 1062 (1990), Spencer et al. Theor Appl Genet 79:625-631(1990)), the hph gene which confers resistance to the antibiotichygromycin (Blochinger & Diggelmann, Mol Cell Biol 4: 2929-2931), andthe dhfr gene, which confers resistance to methotrexate (Bourouis etal., EMBO J. 2(7): 1099-1104 (1983)).

[0140] Transgenic alfalfa plants have been produced using a number ofdifferent genes isolated from both alfalfa and non-alfalfa speciesincluding, but not limited to, the following: the promoter of an earlynodulin gene fused to the reporter gene gusA (Bauer et al., The PlantJournal 10(1):91-105 (1996); the early nodulin gene (Charon et al.,Proc. Natl. Acad. of Sci. USA 94(16):8901-8906 (1997); Bauer et al.,Molecular Plant-Microbe Interactions 10(1):39-49 (1997)); NADH-dependentglutamate synthase (Gantt, The Plant Journal 8(3):345-358 (1995));promoter-gusA fusions for each of three lectin genes (Bauchrowitz etal., The Plant Journal 9(1):31-43 (1996)); the luciferase enzyme of themarine soft coral Renilla reniforms fused to the CaMV promoter(Mayerhofer et al., The Plant Journal 7(6):1031-1038 (1995));Mn-superoxide dismutase cDNA (McKersie et al., Plant Physiology 111(4):1177-1181 (1996)); synthetic crylC genes encoding a Bacillusthuringiensis delta-endotoxin (Strizhov et al., Proc. Natl. Acad. Sci.USA 93(26):15012-15017 (1996)); glucanse (Dixon et al., Gene179(1):61-71 (1996); Masoud et al., Transgenic Research 5(5):313-323));and leaf senescence gene (U.S. Pat. No. 5,689,042).

[0141] Genes successfully transferred into clover using recombinant DNAtechnologies include, but are not limited to, the following: Bt genes(Voisey et al., supra); neomycin phosphotransferase II (Quesbenberry etal., supra); the pea lectin gene (Diaz et al., Plant Physiology109(4):1167-1177 (1995); Eijsden et al., Plant Molecular Biology29(3):431-439 (1995)); the auxin-responsive promoter GH3 (Larkin et al.,Transgenic Research 5(5):325-335 (1996); seed albumin gene fromsunflowers (Khan et al., Transgenic Research 5(3):179-185 (1996)); andgenes encoding the enzymes phosphinothricin acetyl transferase,beta-glucuronidase (GUS) coding for resistance to the Basta® herbicide,neomycin phosphotransferase, and an alpha-amylase inhibitor (Khan etal., supra).

[0142] Other transgenes of interest include, but are not limited to,those coding for or related to lignin content, cellulose content,nitrogen fixation, improved nutrition, color, vitamin content andrecombinantly produced vaccines.

[0143] D. Site-Specific Recombination Systems

[0144] Methods and constructs for targeting of DNA sequences forinsertion into a particular DNA locus, while enabling removal ofrandomly inserted DNA sequences that occur as a by-product oftransformation procedures, are described in U.S. Pat. Nos. 5,527,695 and6,114,600. One manner of removing these random insertions is to utilizea site-specific recombinase system. In general, a site-specificrecombinase system consists of three elements: two pairs of DNA sequence(the site-specific recombination sequences) and a specific enzyme (thesite-specific recombinase). The site-specific recombinase will catalyzea recombination reaction only between two site-specific recombinationsequences.

[0145] A number of different site-specific recombinase systems can beused, including but not limited to the Cre/lox system of bacteriophageP1, the FLP/FRT system of yeast, the Gin recombinase of phage Mu, thePin recombinase of E. coli, and the R/RS system of the pSR1 plasmid. Thetwo preferred site-specific recombinase systems are the bacteriophage P1Cre/10× and the yeast FLP/FRT systems. In these systems a recombinase(Cre or FLP) will interact specifically with its respectivesite-specific recombination sequence (10× or FRT respectively) to invertor excise the intervening sequences. The sequence for each of these twosystems is relatively short (34 bp for 10× and 47 bp for FRT). Currentlythe FLP/FRT system of yeast is the preferred site-specific recombinasesystem since it normally functions in a eukaryotic organism (yeast), andis well characterized. It is thought that the eukaryotic origin of theFLP/FRT system allows the FLP/FRT system to function more efficiently ineukaryotic cells than the prokaryotic site-specific recombinase systems.

[0146] The FLP/FRT recombinase system has been demonstrated to functionefficiently in plant cells. Experiments on the performance of theFLP/FRT system in both maize and rice protoplasts indicates that FRTsite structure, and amount of the FLP protein present, affects excisionactivity. In general, short incomplete FRT sites leads to higheraccumulation of excision products than the complete full-length FRTsites. Site-specific recombination systems can catalyze both intra- andintermolecular reactions in maize protoplasts, indicating that thesystem can be used for DNA excision as well as integration reactions.The recombination reaction is reversible and this reversibility cancompromise the efficiency of the reaction in each direction. Alteringthe structure of the site-specific recombination sequences is oneapproach to remedying this situation. The site-specific recombinationsequence can be mutated in a manner that the product of therecombination reaction is no longer recognized as a substrate for thereverse reaction, thereby stabilizing the integration or excision event.

[0147] E. Vectors

[0148] Expression Units to Express Exogenous DNA in a Plant

[0149] As provided above, several embodiments of the present inventionemploy expression units (or expression vectors or systems) to express anexogenously supplied nucleic acid sequence in a plant. Methods forgenerating expression units/systems/vectors for use in plants are wellknown in the art and can readily be adapted for use in the instantinvention. A skilled artisan can readily use any appropriateplant/vector/expression system in the present methods following theoutline provided herein.

[0150] The expression control elements used to regulate the expressionof the protein can either be the expression control element that isnormally found associated with the coding sequence (homologousexpression element) or can be a heterologous expression control element.A variety of homologous and heterologous expression control elements areknown in the art and can readily be used to make expression units foruse in the present invention. Transcription initiation regions, forexample, can include any of the various opine initiation regions, suchas octopine, mannopine, nopaline and the like that are found in the T1plasmids of Agrobacterium tumafacians. Alternatively, plant viralpromoters can also be used, such as the cauliflower mosaic virus ³⁵Spromoter to control gene expression in a plant. Lastly, plant promoterssuch as prolifera promoter, fruit-specific promoters, Ap3 promoter, heatshock promoters, seed-specific promoters, etc. can also be used. Themost preferred promoters will be most active in male or female gametes.

[0151] Either a gamete-specific promoter, a constitutive promoter (suchas the CaMV or Nos promoter), an organ-specific promoter (such as the E8promoter from tomato) or an inducible promoter is typically ligated tothe protein or antisense encoding region using standard techniques knownin the art. The expression unit may be further optimized by employingsupplemental elements such as transcription terminators and/or enhancerelements.

[0152] Thus, for expression in plants, the expression units willtypically contain, in addition to the protein sequence, a plant promoterregion, a transcription initiation site and a transcription terminationsequence. Unique restriction enzyme sites at the 5′ and 3′ ends of theexpression unit are typically included to allow for easy insertion intoa preexisting vector.

[0153] In the construction of heterologous promoter/structural gene orantisense combinations, the promoter is preferably positioned about thesame distance from the heterologous transcription start site as it isfrom the transcription start site in its natural setting. As is known inthe art, however, some variation in this distance can be accommodatedwithout loss of promoter function.

[0154] In addition to a promoter sequence, the expression cassette canalso contain a transcription termination region downstream of thestructural gene to provide for efficient termination. The terminationregion may be obtained from the same gene as the promoter sequence ormay be obtained from different genes. If the mRNA encoded by thestructural gene is to be efficiently processed, DNA sequences whichdirect polyadenylation of the RNA are also commonly added to the vectorconstruct. Polyadenylation sequences include, but are not limited to theAgrobacterium octopine synthase signal (Gielen et al., EMBO J3:835-846(1984)) or the nopaline synthase signal (Depicker et al., Mol. and Appl.Genet. 1:561-573 (1982)).

[0155] The resulting expression unit is ligated into or otherwiseconstructed to be included in a vector that is appropriate for higherplant transformation. The vector will also typically contain aselectable marker gene by which transformed plant cells can beidentified in culture. Usually, the marker gene will encode antibioticresistance. These markers include resistance to G418, hygromycin,bleornycin, kanamycin, and gentamicin. After transforming the plantcells, those cells having the vector will be identified by their abilityto grow on a medium containing the particular antibiotic. Replicationsequences, of bacterial or viral origin, are generally also included toallow the vector to be cloned in a bacterial or phage host, preferably abroad host range prokaryotic origin of replication is included. Aselectable marker for bacteria should also be included to allowselection of bacterial cells bearing the desired construct. Suitableprokaryotic selectable markers also include resistance to antibioticssuch as ampicillin, kanamycin or tetracycline.

[0156] Other DNA sequences encoding additional functions may also bepresent in the vector, as is known in the art. For instance, in the caseof Agrobacterium transformations, T-DNA sequences will also be includedfor subsequent transfer to plant chromosomes.

[0157] The sequences of the present invention can also be fused tovarious other nucleic acid molecules such as Expressed Sequence Tags(ESTs), epitopes or fluorescent protein markers.

[0158] ESTs are gene fragments, typically 300 to 400 nucleotides inlength, sequenced from the 3′ or 5′ end of complementary-DNA (cDNA)clones. Nearly 30,000 Arabidopsis thaliana ESTs have been produced by aFrench and an American consortium (Delseny et al., FEBS Lett.405(2):129—132 (1997); Arabidopsis thaliana Database,http://genome.www.stanford.edu/Arabidopsis). For a discussion of theanalysis of gene-expression patterns derived from large EST databases,see, e.g., M. R. Fannon, TIBTECH 14:294-298 (1996).

[0159] Biologically compatible fluorescent protein probes, particularlythe self-assembling green fluorescent protein (GFP) from the jellyfishAequorea Victoria, have revolutionized research in cell, molecular anddevelopmental biology because they allow visualization of biochemicalevents in living cells (Murphy et al., Curr. Biol. 7(11):870-876 (1997);Grebenok et al., Plant J. 11(3):573-586 (1997); Pang et al., Plant.Physiol 112(3) (1996); Chiu et al., Curr. Biol. 6(3):325-330 (1996);Plautz et al., Gene 173(1):83-87 (1996); Sheen et al., Plant J.8(5):777-784 (1995)).

[0160] Site-directed mutagenesis has been used to develop a more solubleversion of the codon-modified GFP called soluble-modified GFP (smGFP).When introduced into Arabidopsis, greater fluorescence was observed whencompared to the codon-modified GFP, implying that smGFP is ‘brighter’because more of it is present in a soluble and functional form (Davis etal, Plant Mol. Biol. 36(4):521-528 (1998)). By fusing genes encoding GFPand beta-glucuronidase (GUS), researchers were able to create a set ofbifunctional reporter constructs which are optimized for use intransient and stable expression systems in plants, including Arabidopsis(Quaedvlieg et al., Plant Mol. Biol. 37(4):715-727 (1998)).

[0161] Berger et al. (Dev. Biol. 194(2):226-234 (1998)) report theisolation of a GFP marker line for Arabidopsis hypocotyl epidermalcells. GFP-fusion proteins have been used to localize and characterize anumber of Arabidopsis genes, including geranylgeranyl pyrophosphate(GGPP) (Zhu et al., Plant Mol. Biol. 35(3):331-341 (1997).

[0162] Disabling Genes

[0163] An example of an effective disabling modification would be asingle nucleotide deletion occurring at the beginning of a gene thatwould produce a translational reading frameshift. Such a frameshiftwould disable the gene, resulting in non-expressible gene product andthereby disrupting functional protein production by that gene. If theunmodified gene encodes a protease, for example, protease production bythe gene could be disrupted if the regulatory regions or the codingregions of the protease gene are disrupted.

[0164] In addition to disabling genes by deleting nucleotides, causing atransitional reading frameshift, disabling modifications would also bepossible by other techniques including insertions, substitutions,inversions or transversions of nucleotides within the gene's DNA thatwould effectively prevent the formation of the protein encoded by theDNA.

[0165] It is also within the capabilities of one skilled in the art todisable genes by the use of less specific methods. Examples of lessspecific methods would be the use of chemical mutagens such ashydroxylamine or nitrosoguanidine or the use of radiation mutagens suchas gamma radiation or ultraviolet radiation to randomly mutate genes.Such mutated strains could, by chance, contain disabled genes such thatthe genes were no longer capable of producing functional proteins forany one or more of the domains. The presence of the desired disabledgenes could be detected by routine screening techniques. For furtherguidance, see U.S. Pat. No. 5,759,538.

[0166] Antisense Encoding Vectors

[0167] Methods for inhibiting expression in plants using antisenseconstructs, including generation of antisense sequences in situ aredescribed, for example, in U.S. Pat. Nos. 5,107,065; 5,254,800;5,356,799; 5,728,926; and 6,184,439. The later two patents beingentitled: “Antisense gene systems of pollination control for hybrid seedproduction”.

[0168] Other methods that can be used to inhibit expression of anendogenous gene in a plant may also be used in the present methods. Forexample, formation of a triple helix at an essential region of a duplexgene serves this purpose. The triplex code, permitting design of theproper single stranded participant is also known in the art. (See H. E.Moser, et al., Science 238:645-650 (1987) and M. Cooney, et al., Science241:456-459 (1988)). Regions in the control sequences containingstretches of purine bases are particularly attractive targets. Triplehelix formation along with photocrosslinking is described, e.g., in D.Praseuth, et al., Proc. Nat'l Acad. Sci. USA 85:1,349-1,353 (1988).

[0169] II. Transformation

[0170] A. Plant Transformation

[0171] To introduce a desired gene or set of genes by conventionalmethods requires a sexual cross between two lines, and then repeatedback-crossing between hybrid offspring and one of the parents until aplant with the desired characteristics is obtained. This process,however, is restricted to plants that can sexually hybridize, and genesin addition to the desired gene will be transferred.

[0172] Recombinant DNA techniques allow plant researchers to circumventthese limitations by enabling plant geneticists to identify and clonespecific genes for desirable traits, such as resistance to an insectpest, and to introduce these genes into already useful varieties ofplants. Once the foreign genes have been introduced into a plant, thatplant can than be used in conventional plant breeding schemes (e.g.,pedigree breeding, single-seed-descent breeding schemes, reciprocalrecurrent selection) to produce progeny which also contain the gene ofinterest.

[0173] Genes can be introduced in a site directed fashion usinghomologous recombination. Homologous recombination permits site-specificmodifications in endogenous genes and thus inherited or acquiredmutations may be corrected, and/or novel alterations may be engineeredinto the genome.

[0174] Homologous recombination and site-directed integration in plantsare discussed in U.S. Pat. Nos. 5,451,513; 5,501,967 and 5,527,695.

[0175] B. Transformation Methods

[0176] Methods of producing transgenic plants are well known to those ofordinary skill in the art. Transgenic plants can now be produced by avariety of different transformation methods including, but not limitedto, electroporation; microinjection; microprojectile bombardment, alsoknown as particle acceleration or biolistic bombardment; viral-mediatedtransformation; and Agrobacterium-mediated transformation (see, e.g.,U.S. Pat. Nos. 5,405,765; 5,472,869; 5,538,877; 5,538,880; 5,550,318;5,641,664; 5,736,369 and 5,736,369; Watson et al, Recombinant DNA,Scientific American Books (1992); Hinchee et al., Bio/Tech. 6:915-922(1988); McCabe et al., Bio/Tech. 6:923-926 (1988); Toriyama et al.,Bio/Tech. 6:1072-1074 (1988); Fromm et al., Bio/Tech. 8:833-839 (1990);Mullins et al., Bio/Tech. 8:833-839 (1990); and, Raineri et al.,Bio/Tech. 8:33-38 (1990)).

[0177] Transgenic alfalfa plants have been produced by many of thesemethods including, but not limited to, agrobacterium-mediatedtransformation (Wang et al., Australian Journal of Plant Physiology23(3):265-270 (1996); Hoffman et al., Molecular Plant-MicrobeInteractions 10(3):307-315 (1997); Trieu et al., Plant Cell Reports16:6-11 (1996)) and particle acceleration (U.S. Pat. No. 5,324,646).

[0178] Transformation has also been successfully accomplished in cloverusing agrobacterium-mediated transformation (Voisey et al., BiocontrolScience and Technology 4(4):475-481 (1994); Quesbenberry et al., CropScience 36(4):1045-1048(1996); Khan et al., Plant Physiology105(1):81-88 (1994); Voisey et al., Plant Cell Reports 13(6):309-314(1994)).

[0179] Genetic transformation has also been reported in numerous forageand turfgrass species (Conger B. V. Genetic Transformation of ForageGrasses in Molecular and Cellular Technologies for Forage Improvement,CSSA Special Publication No. 26, Crop Science Society of America, Inc.E. C. Brummer et al. Eds. 1998, pages 49-58). These include orchardgrass(Dactylis glomerata L.), tall fescue (Festuca arundinacea Schreb.) redfescue (Festuca rubra L.), meadow fescue (Festuca pratensis Huds.)perennial ryegrass (Lolium perenne L.) creeping bentgrass (Agrostispalustris Huds.) and redtop (Agrostis alba L.).

[0180] Successful gene transfer in such forages and turfgrasses has beenaccomplished by direct uptake of DNA by protoplasts and by bombardmentof cells or tissues with DNA coated microprojectiles. In both cases, thetransfer is followed by whole plant regeneration. Much of the work hasfocused on developing and improving protocols for the transformation andhave used the reporter gene uidA coding for glucouronidase (GUS) and theselectable marker bar that confers tolerance to phosphinothricin-basedherbicides. Proof of the transformation has been provided by polymerasechain reaction (PCR) techniques, northern hybridization analysis oftranscribed RNA, western blot analysis of soluble protein (geneproduct), and southern blot hybridization of total genomic DNA.

[0181] III. Hemizygosity

[0182] A transgenic plant formed using Agrobacterium transformationmethods typically contains a single gene on one chromosome, althoughmultiple copies are possible. Such transgenic plants can be referred toas being hemizygous for the added gene. A more accurate name for such aplant is an independent segregant, because each transformed plantrepresents a unique T-DNA integration event (U.S. Pat. No. 6,156,953). Atransgene locus is generally characterized by the presence and/orabsence of the transgene. A heterozygous genotype in which one allelecorresponds to the absence of the transgene is also designatedhemizygous (U.S. Pat. No. 6,008,437).

[0183] Assuming normal hemizygosity, selfing will result in maximumgenotypic segregation in the first selfed recombinant generation, alsoknown as the R1 or R1 generation. The R1 generation is produced byselfing the original recombinant line, also known as the R0 or R₀generation. Because each insert acts as a dominant allele, in theabsence of linkage and assuming only one hemizygous insert is requiredfor tolerance expression, one insert would segregate 3:1, two inserts,15:1, three inserts, 63:1, etc. Therefore, relatively few R1 plants needto be grown to find at least one resistance phenotype (U.S. Pat. Nos.5,436,175 and 5,776,760).

[0184] As mentioned above, self-pollination of a hemizygous transgenicregenerated plant should produce progeny equivalent to an F2 in whichapproximately 25% should be homozygous transgenic plants.Self-pollination and testcrossing of the F2 progeny to non-transformedcontrol plants can be used to identify homozygous transgenic plants andto maintain the line. If the progeny initially obtained for aregenerated plant were from cross pollination, then identification ofhomozygous transgenic plants will require an additional generation ofself-pollination (U.S. Pat. No. 5,545,545).

[0185] IV. Semi-Sterility and Genetic Sterility Filter

[0186] A. The Gametophytic Sterility Trait (GST)

[0187] The GST can be composed of two or three elements: a sex-specificpromoter, a suicide gene and, optionally, a region encoding a transposonand/or transposase. Normally, the GST construct ends in a transcriptionterminator element. The inclusion of a transposon or a transposasesource is specific to the application of selecting for dispersedtranspositions and not necessarily used for the purposes of eliminatingtransmission of transgenes.

[0188] Sex-specific promoters that may be used include but are notlimited to: pollen-specific promoters from maize, rice, tomato, tobacco,Arabidopsis and Brassica. Several other examples can be found inGenBank. The promoter must be specific to one sex (male or female) andspecifically drive gene expression after meiosis I when homologouschromosomes have separated into different cells.

[0189] The suicide gene is used to kill unwanted meiotic products.Suicide genes include but are not limited to: barnase, tasselseed2 andthe diphtheria toxin A gene. Two alternatives to using a suicide geneinclude 1) using antisense RNA technology to inhibit expression of genesessential to the viability of the pollen or the egg; or 2) mutationsthat are incapable of transmission through one of the sexes, such asdeletions that are not pollen-transmitted. Both of these alternativescan be used to achieve semi-sterility.

[0190] B. Semi-Sterility and Genetic Sterility Filter

[0191] The terms “semi-sterility” and “genetic sterility filter” areused by the inventors to convey the idea that since suicide geneexpression is confined to post-meiosis I, only 50% of gametes will beeliminated when the GST locus is hemizygous and segregates normally inmeiosis. This is due to the fact that when the GST locus is present inthe genome in a single copy (hemizygous condition), the suicide genewill be transmitted to approximately one-half of the products ofmeiosis, resulting in a 50% sterility rate. Pollen inheriting the GSTwill not survive if a pollen specific promoter is operably linked to thesuicide gene.

[0192] The production of 50% viable pollen is necessary for the recoveryof dispersed transpositions and/or to prevent transgene transmissionwithout a major effect on male fertility.

[0193] C. Gametophytic Semi-Sterility

[0194] The current invention describes the use of a technology thatutilizes gametophytic “semi-sterility”, such as pollen semi-sterility,to generate a “genetic sterility filter” that eliminates gametes thatinherit a specific transgene complex. Incorporation of a pollen-specificpromoter into the GST prevents the transmission of transgenes linked tothe GST in pollen inheriting this transgene complex.

[0195] This transgene complex may also contain a launching site for atransposable element and/or transposase gene. In this case, theelimination of the transgene complex, along with the transposon donorsite and/or transposase, has the net effect of eliminating nearbytranspositions while enriching for transposition events that haverecombined with the transgene complex or that are dispersed (no longerlinked to the GST) throughout a genome. This methodology overcomesseveral current limitations of transposon mutagenesis strategies thatfavors mostly localized over dispersed transpositions. The inventiongreatly improves on the current use of negative selectable markers toachieve transposon dispersion (Sundaresan, V., et al., (1995) Genes Dev.9/14:1797-810; Tissier, A. F. et al., (1999) The Plant Cell Vol.11:1841-1852). Moreover, the use of the genetic sterility filter toeliminate transmission of a transposase source stabilizes the newlytransposed elements in progeny, thereby eliminating somatic or secondarytransposition events that hamper mutation identification.

[0196] The nature of the semi-sterility trait and its associatedtransposon and/or transposase, may differ in details depending on thechoice of suicide genes, promoters, transposon systems, and species. Itis emphasized, however, that the same basic technology of semi-sterilitycan be used to recover transpositions in many plants, both monocots anddicots, in such species such as maize, rice, soybeans, wheat, oats,barley, and in non-plant systems, such as animals and fungi, that can besexually propagated. Furthermore, an alternative strategy to microsporeelimination is to eliminate female transmission of the transgene complexby engineering a megaspore suicide trait.

[0197] The semi-sterility trait is used to eliminate the products ofmeiosis (gametes) that carry a particular chromosomal region, such as atransposon launching site and/or transposase gene. This “geneticsterility filter” is used to eliminate male or female transmission of atransgene complex. This elimination process has the net effect ofenriching for unlinked (dispersed) transposed elements or elements thatrecombined from the launching site; it is also used to simultaneouslyeliminate transmission of other genes, such as a transposage gene,thereby stabilizing transpositions in progeny. To achievesemi-sterility, a number of preferred methods are contemplated by theinstant invention. One method relies on directing microspore-specificexpression of a suicide gene to kill unwanted microspores. This methodis achieved by employing a specific promoter, such as a pollen-specificpromoter, fused to an appropriate suicide gene, thereby killing only theproducts of meiosis that have inherited the gene fusion. For a singlecopy transgene in hemizygous condition, this represents 50% of thegametes. Because pollen is produced in large excess, reducing pollenfertility by 50% has no major consequence on subsequent seed production.

[0198] The aspect of the current invention relating to generatingsemi-sterility differs from previous methods aimed at achieving fullmale-sterility for hybrid seed production that are known in the art. Inthese methods, the aim has been to achieve complete male sterility tofacilitate the commercial production of hybrid seed. This usuallyinvolves expression of the suicide gene in the sporophyte or in allmicrospores. For instance, bamase expression in tapetal cells results incomplete male sterility (Beals, T. P. et al. (1997) The Plant Cell. Vol.9:1527-45). In hybrid seed production, pollen semi-sterility would beinsufficient to achieve the desired result, i.e. outcrossing ofmale-sterile (female) parent. In cases where a microspore-specificsuicide gene and its associated elements are present in the genome in asingle copy (in hemizygous condition), the suicide gene will betransmitted to approximately one-half of the products of meiosis,resulting in an average of 50% semi-sterility, a rate of viable pollenproduction that is commercially unacceptable for hybrid seed production.For the present invention, the production of 50% viable pollen isnecessary and important to recover dispersed transpositions and/or toprevent transposase transmission. Hence, the gametophytic sterilitytrait is used as a filter to eliminate undesirable genomes while at thesame time allowing other genomes (non-transgenic and genomes containingtransposed elements without the donor element and/or transposase genepresent) to be transmitted. In one embodiment, the present inventionmakes us of this “genetic sterility filter” to eliminate a transgenecomplex containing a transposon launching site and/or transposasesource.

[0199] V. Breeding Methods

[0200] Open-Pollinated Populations. The improvement of open-pollinatedpopulations of such crops as rye, many maizes and sugar beets, herbagegrasses, legumes such as alfalfa and clover, and tropical tree cropssuch as cacao, coconuts, oil palm and some rubber, depends essentiallyupon changing gene-frequencies towards fixation of favorable alleleswhile maintaining a high (but far from maximal) degree ofheterozygosity. Uniformity in such populations is impossible andtrueness-to-type in an open-pollinated variety is a statistical featureof the population as a whole, not a characteristic of individual plants.Thus, the heterogeneity of open-pollinated populations contrasts withthe homogeneity (or virtually so) of inbred lines, clones and hybrids.

[0201] Population improvement methods fall naturally into two groups,those based on purely phenotypic selection, normally called massselection, and those based on selection with progeny testing.Interpopulation improvement utilizes the concept of open breedingpopulations; allowing genes for flow from one population to another.Plants in one population (cultivar, strain, ecotype, or any germplasmsource) are crossed either naturally (e.g., by wind) or by hand or bybees (commonly Apis mellifera L. or Megachile rotundata F.) with plantsfrom other populations. Selection is applied to improve one (orsometimes both) population(s) by isolating plants with desirable traitsfrom both sources.

[0202] There are basically two primary methods of open-pollinatedpopulation improvement. First, there is the situation in which apopulation is changed en masse by a chosen selection procedure. Theoutcome is an improved population that is indefinitely propagable byrandom-mating within itself in isolation. Second, the synthetic varietyattains the same end result as population improvement but is not itselfpropagable as such; it has to be reconstructed from parental lines orclones. These plant breeding procedures for improving open-pollinatedpopulations are well known to those skilled in the art and comprehensivereviews of breeding procedures routinely used for improvingcross-pollinated plants are provided in numerous texts and articles,including: Allard, Principles of Plant Breeding, John Wiley & Sons, Inc.(1960); Simmonds, Principles of Crop Improvement, Longman Group Limited(1979); Hallauer and Miranda, Quantitative Genetics in Maize Breeding,Iowa State University Press (1981); and, Jensen, Plant BreedingMethodology, John Wiley & Sons, Inc. (1988).

[0203] Mass Selection. In mass selection, desirable individual plantsare chosen, harvested, and the seed composited without progeny testingto produce the following generation. Since selection is based on thematernal parent only, and there is no control over pollination, massselection amounts to a form of random mating with selection. As statedabove, the purpose of mass selection is to increase the proportion ofsuperior genotypes in the population.

[0204] Synthetics. A synthetic variety is produced by crossing inter sea number of genotypes selected for good combining ability in allpossible hybrid combinations, with subsequent maintenance of the varietyby open pollination. Whether parents are (more or less inbred)seed-propagated lines, as in some sugar beet and beans (Vicia) orclones, as in herbage grasses, clovers and alfalfa, makes no differencein principle. Parents—are selected on general combining ability,sometimes by test crosses or topcrosses, more generally by polycrosses.Parental seed lines may be deliberately inbred (e.g. by selfing or sibcrossing). However, even if the parents are not deliberately inbred,selection within lines during line maintenance will ensure that someinbreeding occurs. Clonal parents will, of course, remain unchanged andhighly heterozygous.

[0205] Whether a synthetic can go straight from the parental seedproduction plot to the farmer or must first undergo one or two cycles ofmultiplication depends on seed production and the scale of demand forseed. In practice, grasses and clovers are generally multiplied once ortwice and are thus considerably removed from the original synthetic.

[0206] While mass selection is sometimes used, progeny testing isgenerally pretferred for polycrosses, because of their operationalsimplicity and obvious relevance to the objective, namely exploitationof general combining ability in a synthetic.

[0207] The number of parental lines or clones that enter a syntheticvary widely. In practice, numbers of parental lines range from 10 toseveral hundred, with 100-200 being the average. Broad based syntheticsformed from 100 or more clones would be expected to be more stableduring seed multiplication than narrow based synthetics.

[0208] Hybrids. A hybrid is an individual plant resulting from a crossbetween parents of differing genotypes. Commercial hybrids are now usedextensively in many crops, including corn (maize), sorghum, sugarbeet,sunflower and broccoli. Hybrids can be formed in a number of differentways, including by crossing two parents directly (single cross hybrids),by crossing a single cross hybrid with another parent (three-way ortriple cross hybrids), or by crossing two different hybrids (four-way ordouble cross hybrids).

[0209] Strictly speaking, most individuals in an out breeding (i.e.,open-pollinated) population are hybrids, but the term is usuallyreserved for cases in which the parents are individuals whose genomesare sufficiently distinct for them to be recognized as different speciesor subspecies. Hybrids may be fertile or sterile depending onqualitative and/or quantitative differences in the genomes of the twoparents. Heterosis, or hybrid vigor, is usually associated withincreased heterozygosity that results in increased vigor of growth,survival, and fertility of hybrids as compared with the parental linesthat were used to form the hybrid. Maximum heterosis is usually achievedby crossing two genetically different, highly inbred lines.

[0210] The production of hybrids is a well-developed industry, involvingthe isolated production of both the parental lines and the hybrids whichresult from crossing those lines. For a detailed discussion of thehybrid production process, see, e.g., Wright, Commercial Hybrid SeedProduction 8:161-176, In Hybridization of Corp Plants, supra.

[0211] VI. Transposons and Transposable Elements

[0212] Transposons are genetic elements capable of transposition(movement) from a donor chromosomal site to a target site on the samechromosome or different chromosome. Transposons cause mutations byinsertion into coding sequences, introns, and promoters, oftencompletely eliminating target gene activity. Mutations caused bytransposons can often be destabilized by subsequent excision of thetransposon from the gene. One or more proteins, collectively referred toas “transposase”, are required for the excision and integration of thetransposon. Transposons that encode their own source of transposase arereferred to as autonomous elements. Supplying transposase in trans cantranspose transposons that do not encode transposase but containterminal sequences, usually inverted repeats, required for transposition(non-autonomous elements).

[0213] A. Ds Elements

[0214] In one of the preferred embodiments, a Ds element is contemplatedsuch that it contains the terminal sequences required for transposition(Coupland, G., et al., (1989) Proc Natl Acad Sci USA 86:9385-8;Chatterjee, S. et al., (1995) Mol Gen Genet. 249:281-8), a minimalpromoter (−47 CaMV ³⁵S promoter) fused to GUS reporter gene for enhancerdetection; in opposite orientation, a splice acceptor site fused to anenhanced fluorescent, dual-spectrum GFP gene (Haseloff, J., et al.,(1997) Proc Natl Acad Sci USA. 94:2122-7; Haseloff, J., et al., (1999)Methods Mol. Biol. 122:241-59; Haseloff, J. (1999) Methods Cell Biol.58:139-51) for gene trapping purposes. This Ds element can be used toscreen for enhancer traps in one insertion orientation or gene traps inthe other orientation, with respect to target gene transcription. Asecond embodiment using Ds substitutes a transcriptional activator atone end of the Ds element in order to generate gain-of-functionmutations. Both elements contain a lox P site for site-specificrecombination (Osborne, B. I., et al., (i995) 7:687-701; Medberry, S.L., et al., (1995) Nucleic Acids Res. 23:485-90; Qin, M., et al., (1994)Proc Natl Acad Sci USA 91:1706-10) and the bar gene Thompson, C. J., etal., (1986) EMBO J. 6:2519-23) for herbicide (Finale®) for tissueculture and soil selection. Finale is the registered trademark of aglufosinate herbicide.

[0215] B. Utility of Transposon and Insertion Mutations in Plants

[0216] Transposons have great utility in genetic analysis and functionalgenomic analysis of bacterial, fungal, plant and animal genomes. Inplants, genetically engineered transposons have been successfullyintroduced into several species (for review see Sundaresan, V, (1996)Trends Plant Sci. Vol. 1:184-190) including rice (Izawa, T. et al.,(1997) Plant Mol Biol Vol. 35/1-2:219-29), tobacco, Arabidopsis, lettuceand several others. Several investigators have come up with ingeniousmethods to enhance the efficiency of recovering transposition events,including methods for positive selections for transposition (Fedoroff,N. V. et al., (1993) Plant J. Vol.:3:273-289; Honma, M. A. et al.,(1993) Proc Natl Acad Sci USA Vol. 90/13:6242-6), selection for unlinkedelements, (Tissier et al., (1999) ThePlant Cell Vol. 11:1841-1852; W.R.; Sundaresan, et al., (1995) Genes Dev. 9/14:1797-810), dispersedlaunching sites throughout a genome (Cooley, M. B. et al., (1996) MolGen Genet Vol. 252/1-2:184-94, Knapp, S. et al., (1994) Mol Gen GenetVol. 243/6:666-73; Osborne, B. I. et al., (1991) Genetics Vol.129/3:833-44; Takken, F. L. et al., (1998) Plant J Vol. 14/4:401-11,Thomas, C. M, et al., (1994) Mol Gen Genet Vol. 242/5:573-85; van derBiezen, E. A. et al., 1996 Mol Gen Genet Vol. 251/3:267-80) and addingfeatures into elements such as enhancer and gene traps (for reviews seeSharknes, W. C., (1990) Biotechnology 8:827-831; W. R.; Sundaresan, etal., (1995) Genes Dev 9/14:1797-810) and transcriptional activation(Fritze, K. et al. (1995) Methods Mol Biol Vol. 44:281-94; Kakimoto, T.,(1996) Science Vol. 274/5289:982-5; Kardailsky, I.; et al., (1999)Science Vol. 286/5446:1962-5).

[0217] Even given the enormous progress in the utility of transposons,they still have limitations for functional genomic analysis.Transpositions are often intrachromosomal, often within a short physicaldistance between donor and target sites (Moreno, M. A et al., (1992)Genetics Vol. 131:939-956; Athma, P. et al., (1992) Genetics Vol.131:199-209). This limitation means that most new insertions occur inregions surrounding the donor site (launching site) with many fewerelements found dispersed randomly throughout the genome. Secondly, theprocess of transposition is not usually controlled leading to a greatmany somatic insertions, which are not transmitted to progeny, anddevelopmentally early transpositions that can lead to non-concordance ingerminally transmitted events. The inability to control transpositionssomatically and temporally can result in a high background of falsepositive insertions in genes of interest due to the non-correspondencebetween somatic and germinal mutational events.

[0218] C. Current State-of-the-Art Methods to Recover DispersedTranspositions

[0219] Two methods are currently available to partially overcome thelimitation of non-dispersed transpositions. One method makes use offirst dispersing transposon launching sites throughout a genome. Thismethod, however, requires a large number of transgenic starter lines toachieve widespread genome coverage. A second method to dispersetransposition events is to employ a negative selectable marker(s), suchas iaaH, peha, R404, or a cytosine deaminase gene, to select against thedonor site containing the transposon and/or transposase. Selectionagainst the donor element also selects against nearby (linked)transpositions resulting in enrichment for unlinked transpositions.Negative selections have been used in Arabidopsis to recover unlinked Dsand Spm transpositions (W. R.; Sundaresan, et al., (1995) Genes Dev.9/14:1797-810; Tissier, A. F., et al., (1999) The Plant Cell Vol.11:1841-1852) and two negative selectable markers are based onproherbicide conversion (O'keefe, D. P. et al., (1994) Plant PhysiolVol. 105:473-482; Dotson, S. B. et al., (1996) The Plant Journal Vol.10/2:383-392), a process theoretically amenable to soil selections.

[0220] Nevertheless, the use of negative selectable markers imposesserious limitations on the recovery of large numbers of independenttranspositions. First, several of these markers require the use oftissue-culture based (W. R.; Sundaresan, et al., (1995) Genes Dev.9/14:1797-810; transposition (Fedoroff, N. V. et al., (1993) Plant J.Vol.:3:273-289), a labor-intensive procedure that adds great expense andtime to the process of recovering large numbers of dispersedtranspositions. Second, negative selections are based on the eliminationof progeny carrying linked elements or transposase, or both, by chemical(Tissier, A. F., et al., (1999) The Plant Cell Vol. 11:1841-1852; W. R.;Sundaresan, et al., (1995) Genes Dev. 9/14:1797-810). Progenyelimination can be problematic when seed number is limited. Forinstance, in a plant such as rice (Orza sativa), a predominantlyself-pollinating species, outcrossing is tedious and time-consuming,greatly limiting the number of progeny that can be readily obtained.Hence, if transposition rates are low and unlinked transpositionsrepresent the minority of transposition events, it would be costly andimpractical to use negative selections to recover dispersedtranspositions. For instance, accounting for a rate of 1-5%transposition, 50% meiotic segregation, and only 30% unlinkedtranspositions, to recover 10,000 transpositions by outcrossing wouldrequire an estimated several million progeny plants. Finally, the use ofnegative selectable agents, such as proherbicides, can have a seriousenvironmental impact and be costly due to the applications of chemicalsthat may be needed to select for large numbers of progeny carryingdispersed transpositions. Moreover, most of these chemicals are notapproved for field use.

[0221] VII. Transposase

[0222] Florally Expressed Transposase

[0223] A further limitation associated with using transposons formutagenesis, gene-tagging and functional genomic analysis, is the lackof developmental and temporal control over the transposition process. Inmost cases transposase gene expression, under the control of its ownpromoter, or under the control of constitutive promoters, occurs duringvegetative development of the plant. Vegetative expression of atransposase source leads to somatic transpositions. These transpositionsare transmitted to progeny (germinal transpositions) only when thesesomatic cell lineages later produce megaspores or microspores. This isproblematic for two reasons. One reason is that transpositions thatoccur during vegetative or early reproductive development can beclonally propagated and later transmitted into many gametes, resultingin a large number of non-independent elements recovered in progeny. Thisis undesirable when large numbers of independent transpositions areneeded for functional genomic analysis or gene-tagging. Second, somatictranspositions often do not include lineages that give rise to gametes,such as those occurring in epidermal lineages or in terminal vegetativestructures. These transpositions are never meiotically transmitted andtherefore go unrecovered in progeny. This is problematic because thesesomatic events can be falsely identified in tissue DNA samples asmutations, yet are never recovered in progeny. This is especiallyproblematic when mutational screens are PCR-based examples chemical(Tissier, A. F., et al., (1999) The Plant Cell Vol. 11:1841-1852;McKinney, E. C., et al., (1995) Plant J. Vol. 8:613-622; Krysan, P. J etal., (1996) Proc. Natl. Acad. Sci. USA Vol. 93:8145-8150; Frey, M etal., (1997) Science Vol. 277:696-699).

[0224] In a further embodiment of the instant invention, to minimize theproblem of somatic transpositions, a transposase gene is placed underthe control of a floral-specific promoter that drives gene expression insubepidermal lineages of the flower that lead to the production ofmicrospores and megaspores. Such promoters include those found in genessuch as agamous, apetala1 apetala2, apetala3, pistillate, and theirhomologs found in other plant species such as maize, and rice. Forexample, the apetala3 promoter drives expression of a reporter gene inpetal and sepal primordial cells of the developing floral meristems.Transposase expression under the control of the ap3 promoter results intranspositions that are confined to floral development and, whenoccurring in lineages that give rise to microspores, these events willbe transmitted to the next generation. Control of transposition by thismethod has two effects: 1) it shuts down somatic transpositions; and 2)it leads to a large number of independent transpositions when pollen isderived from many different floral meristems. Hence, somatic tissue canbe sampled without the concern of somatic or secondary transpositions,and each floral meristem becomes an independent source of transpositionevents.

[0225] VIII. Rice—A Model Plant System

[0226] A. Rice Agriculture in the US and Worldwide

[0227] Approximately half of the world's population derives its caloricintake mainly from rice. Annual worldwide production levels are over 400million metric tons, grown on over 200 million hectares (Anonymous(2000) USDA World Agriculture Supply and Demand Estimates. USDAAgricultural Marketing Service. Publication WASDE-362).

[0228] At present, the U.S. produces 7.5 million metric tons of rice peryear, planted on 1.4 million hectares, resulting in $1.7 billion dollarsin commerce (Anonymous (2000) USDA World Agriculture Supply and DemandEstimates. USDA Agricultural Marketing Service. Publication WASDE-362).More than two-thirds of the rice produced in this country is exported tomarkets, mainly in Asia and Latin America, making the U.S. the thirdlargest exporter worldwide.

[0229] Approximately 99% of the rice varieties currently grown are theresult of public breeding programs, many originating from breedingprograms sponsored by CGIAR international research centers(http://www.cgiar.org/irri/crucial.htm) such as International RiceResearch Institute (IRR1) and International Center for TropicalAgriculture (CIAT). The majority of U.S. rice varieties are developed inthe states of Arkansas, Louisiana, Mississippi, Texas and California.Agricultural biotechnology is becoming increasingly important to developmodern varietal rice lines; biotechnology development is expected togreatly assist the U.S. rice farmers competing in this globalmarketplace

[0230] B. Rice as a Model System for Monocot Development

[0231] Cereals include the most important food crops in the world, andare considered a relatively recent taxon, evolving from a commonancestor only 65 million years ago (Martin, W., et al., (1989) Nature339:46-48; Moore, G., et al., (1995) Trends Genet. 11:81-82). This younghistory is reflected in a remarkable degree of conservation in genestructure and order even though differences have arisen in genome size,haploid chromosome number, and variations in repetitive sequencecomposition (Moore, G., et al., (1993) Bio/technology. 11:584-589). Forexample, the maize genome is 8-fold larger than that of rice (Ahn, S. etal., (1993) Genetics 90:7980-7984) and organized into a different numberof chromosomes, yet comparative molecular analysis has shown thatextensive synteny can be identified between much of their genomes (Ahn,S. et al., (1993) Genetics. 90:7980-7984; Bennetzen, J. L. et al.,(1993) Trends Genet. 9:259-261).

[0232] Rice is an outstanding model plant for the cereal grasses. Ricecan be used to investigate basic biological issues and to learn aboutagronomic traits such as yield, hybrid vigor, and single and multigenicdisease resistance. Different races of rice are adapted to a widevariety of environmental situations, from tropical flooding to temperatedry land, so it is a model for real life adaptive responses.

[0233] Rice has a relatively short generation time (90-120 days), makingit possible to obtain three or more generations per year. A largecollection of mutations have been discovered and characterized in rice.

[0234] Transgenic rice is efficiently generated by eitherAgrobacterium-mediated (Hiei, Y., et al., (1994) Plant J. 6:271-82;Hiei, Y., et al., (1997) Plant Molec Bio. 35:205-218; Zhang, J., et al.,(1997) Mol Biotechnol. 8:223-31) or biolistic methods (Christou, P., etal., (1991) Biotechnology. 9:957-962; Buchholz, W. G., et al., (1998)Methods Mol. Biol. 81:383-96). Most importantly, rice has a genome sizeapproximately 500 megabases (Mb) (Arumanagathan, K. et al., (1991) PlantMol. Biol. Report. 9:208-218), only about 3-fold larger than that of theArabidopsis genome, and scheduled to be sequenced around 2004. As amember of the Graminae and an important crop plant, a wealth offundamental information about important aspects of plant biology can belearned from the rice genomics (McCouch, S. (1998) Proc. Natl. Acad.Sci. USA 95:1983-5; Wilson, W. A., et al., (1999) Genetics. 153:453-73).

[0235] C. Rice Genomics

[0236] Rice is one of the most densely mapped plant genomes (McCouch, S.R., et al., (1997) Plant Mol. Biol. 35:89-99; Panaud, O., et al., (1996)Mol Gen Genet. 252:597-607). The two best-developed recombinational mapsare those developed at Cornell (http://genome.cornell.edu/rice/) and atthe Rice Genome Project (RGP) in Japan (http://www.dna.affrc.go.jp), onwhich more than 3,000 RFLP and SSR markers have been mapped. TheYAC-based physical map of rice covers more than 64% of the genome andcontains 4,000 mapped ESTs (Ashikawa, I., et al., (1999) Genome.42:330-7). A PAC library of 71,000 clones has been mapped with STSs andESTs and the mapped clones cover approximately 30% of the genome. TwoBAC libraries with 37,000 and 55,000 members have been BAC-end sequencedand finger printed and a BAC-based physical map has been constructed(http://www.genome.clemson.edu/projects/rice/).

[0237] The rice genome is estimated to contain 500 Mb (Arumanagathan, K.et al., (1991) Plant Mol. Biol. Report. 9:208-218) and 340,000 genes.The International Rice Genome Sequencing Project was formed in 1998 toobtain the complete genome sequence of Oryza sativa ssp. japonica cv.Nipponbare. Ten countries are collaborating in this effort. Currentlyabout 10 Mb have been submitted to GenBank.

[0238] D. Utility of a Transposon-Based Genomics Program

[0239] The main justification for the use of transposons is theirdistinct advantage over other types of mutagens for functional genomicstudies. In rice, T-DNA and retrotransposon mutagenesis have seriouslimitations. Currently, both methods require continual tissue cultureselection and somatic regeneration to recover insertions, processes thatare inefficient, time-consuming and prone to induction of somaclonalvariation (Bao, P. H., et al. (1996) Transgenic Res. 5:97-103; Evans, D.A. (1989) Trends Genet. 5:46-50). Both agents generate only stableinsertions, which subsequently limit the utility of any mutant allele.To further complicate matters, T-DNA insertions are often large, complextandem arrays, causing difficulties with molecular analysis of mutantalleles (McKinney, E. C., et al., (1995) Plant J. 8:613-622; Krysan, P.J., et al., (1996) Proc. Natl. Acad. Sci. USA 93:8145-8150; Krysan, P.J., et al., (1999) Plant Cell. 11:2283-2290; Galbiati, et al., (2000)Functional & Integrative Genomics, in press).

[0240] In many instances, single loss-of-function mutations, such asT-DNA- or retrotransposon-induced alleles, will not provide sufficientinformation to derive gene function. For instance, based on limitedstudies to date, many gene disruptions do not produce a readilydiscernable phenotype (McKinney, E. C., et al., (1995) Plant J.8:613-622; Krysan, P. J., et al., (1996) Proc. Natl. Acad. Sci. USA93:8145-8150). This does not imply lack of gene importance, however,since many of these genes will have partially redundant, overlapping orspecific functions not detectable based on morphological ordevelopmental screening of mutant lines. More detailed information, suchas expression analysis and additional alleles, will be necessary.

[0241] On the other hand, two-element transposon mutagenesis, such asthe AciDs system, can generate stable gene disruptions by simpleinsertion. Genetically engineered Ds elements have been successfullyintroduced into several plant species (for review see Martienssen, R. A.(1998) Proc. Natl. Acad. Sci. USA 95:2021-6; Sundaresan, V. (1996)Trends Plant Sci. 1:184-190) including rice (Izawa, T., et al. (1997)Plant Mol. Biol. 35:219-29).

[0242] The utility of transposons for functional genomics has beengreatly enhanced by building features into synthetic transposons such asenhancer and gene traps (for reviews see Martienssen, R. A. (1998) Proc.Natl. Acad. Sci. USA 95:2021-6; Sharknes, W. C. (1990) Biotechnology8:827-831; Sundaresan, V., Springer, et al., (1995) Genes Dev.9:1797-810) and transcriptional activation (Fritze, K. et al., (1995)Methods Mol. Biol. 44:281-94; Kakimoto, T. (1996) Science. 274:982-5;Kardailsky, I., et al., (1999) Science. 286:1962-5). By incorporatingsuch features, even genes that are genetically redundant can befunctionally analyzed. Most importantly, the ability to remobilize atransposon creates the unique opportunity to efficiently generatederivative alleles and to efficiently mutate nearby genes by localizedtransposition properties of these elements (Long, D., et al., (1997)Plant J. 11:145-8; Jones, J. D. G., et al., (1990) Plant Cell.2:701-707; Osborne, B. I., et al., (1991) Genetics. 129:833-44).Starting with a just a few, well-characterized transgenic lines and theappropriate genetic strategies, an extensive collection of dispersedtranspositions can be efficiently generated without the need forsubsequent tissue culture selection or regeneration.

[0243] E. Randomly Dispersing Ds Throughout the Rice Genome

[0244] The present invention includes methods that may be applied tospecific genomes, including but not limited to, the rice genome. Rice isan outstanding model plant for the cereal grasses. In applying themethods of the instant invention to the rice genome, one goal is toproduce an extensive collection of stable Ds insertions that aredistributed throughout the rice genome. To accomplish this task, severalgenetic strategies are contemplated, with the main goals of minimizingnon-independent transpositions, dispersed Ds transpositions throughoutthe rice genome, and stabilized transposed elements in progeny. Aspecific strain contemplated for use in the methods of the instantinvention is Oryza sativa ssp. japonica cv. Nipponbare, the strain beingsequenced by the IRGSP.

[0245] In one embodiment of the instant invention, randomly dispersingDs throughout the rice genome is contemplated. Because Ds tends totranspose locally often over short genetic distances, genetic strategiesmust be used to counter this bias. In the past, this has beenaccomplished using various methods that basically involve selectingagainst the Ds launching site in progeny (Sundaresan, V., et al., (1995)Genes Dev. 9:1797-810; Tissier, A. F. et al., (1999) The Plant Cell11:1841-1852), or by initially dispersing many launching sitesthroughout a genome (Osborne, B. I., et al., (1991) Genetics.129:833-44; Cooley, M. B., et al., (1996) Mol Gen Genet. 252:184-94;Knapp, S., et al., (1994) Mol Gen Genet. 243:666-73; Takken, F. L., etal., (1998) Plant J. 14:401-11; Thomas, C. M., et al., (1994) Mol GenGenet. 242:573-85; van derBiezen, E. A., et al., (1996) Mol Gen Genet.251:267-80).

[0246] Two strategies for dispersing Ds transpositions are contemplated.The first method is broadly directed to including a pollen-specificsuicide trait gene on the Ds launching site to eliminate transmission ofthe launching site along with any linked transposed elements. Thesuicide trait is engineered by incorporating a pollen-specific promoterdriving expression of an appropriate cell death gene. Since thelaunching site construct is single copy and hemizygous in stock plants,50% of the products of meiosis inherit T-DNA and undergo geneticsuicide; the remaining products produce viable pollen. Those transposedDs that have recombined, either intra- or inter-chromosomally with thelaunching site, are pollen transmitted. These elements are readilydetected in progeny by incorporating an herbicide marker (bar gene) intothe Ds element. The herbicide marker serves a dual purpose—as an initialtissue-culture selectable marker for rice transformation, and later as asoil-based selection for progeny harboring unlinked Ds elements.

[0247] The pollen suicide method has distinct advantages over previousstrategies to select for unlinked transpositions. Its main advantage isthat pollen is produced in vast excess and pollen semi-sterility isenvironmentally sound and will have little, if any, impact on seedproduction. In progeny, 50% of the testcross offspring (or 75% whenselfing) are culled by negative selection simply because they inheritthe launching site (and/or transposase gene, as explained below). Inrice, when outcrossing is required to recover transpositions, seedproduction can be a limiting factor. Moreover, chemicals, such asproherbicides used for negative selection (Tissier, A. F., et al.,(1999) The Plant Cell. 11:1841-1852; Dotson, S. B., et al., (1996) ThePlant Journal. 10:383-392) are neither commercially available norfederally approved for field use. Tissue culture based negativeselections (Sundaresan, V., et al., (1995) Genes Dev. 9:1797-810;Kobayashi, T., et al., (1995) Jpn J. Genet. 70:409-22) are impracticalin rice.

[0248] In one embodiment of the instant invention, the pollen-specificsuicide trait is engineered using an appropriate promoter driving theexpression of a suicide gene. Several suicide genes are available,including the barnase gene (Goldman, M. H., et al., (1994) EMBO J.13:2976-84), related RNases (Fedorova, N. D., et al., (1994) Mol Biol(Mosk). 28:468-71), diphtheria toxin A chain gene (Tsugeki, R. et al.,(1999) Proc Natl Acad Sci USA. 96:12941-6; Nilsson, O., et al., (1998)Plant J. 15:799-804; Uk Kim, et al., (1998) Mol Cells. 8:310-7; Day, C.D., et al., (1995) Development 121:2887-95) and others (DeLong, A., etal., (1993) Cell. 74:757-768). Use of the barnase gene has been shown tobe an effective way to generate microspore-autonomous cell death whenfused to a pollen-specific promoter (Custers, J. B., et al., (1997)Plant Mol. Biol. 35:689-99). In contrast to previous methods, themethods of the instant invention depend on generating semi-sterility, asopposed to complete male sterility, which may be achieved in one aspectof the present invention by engineering barnase expression specificallyin microspores. Several promoters are available for this purpose,including both rice (Zou, J. T., et al., (1994) Amer. J. Botany.81:552-561) and maize pollen-specific promoters (Hamilton, D. A., etal., (1992) Plant Mol. Biol. 18:211-8), and pollen-specific promotersfrom several dicotyledonous species (Twell, D., et al., (1991) GenesDev. 5:496-507; Kulikauskas, R. et al., (1997) Plant Mol. Biol.34:809-14; Custers, J. B., et al., (1997) Plant Mol. Biol. 35:689-99;Albani, D., et al., (1991) Plant Mol. Biol. 16:501-13; Kim, Y. et al.,(1992) Transgenic Res. 1:188-94; Twell, D., et al.; (1990) Development.109:705-13; van Tunen, A. J., et al., (1990) Plant Cell. 2:393-401). Ina preferred embodiment, a heterologous pollen-specific promoter, such asthe maize promoter (Hamilton, D. A., et al., (1992) Plant Mol. Biol.18:211-8), is contemplated to minimize the possibility of genesilencing.

[0249] Evaluation of the effectiveness of the pollen suicide mechanismin eliminating T-DNA (launching sites), may be achieved for example, byemploying a construct containing the suicide gene and the bar genetransformed into an organism such as rice via Agrobacterium-mediatedT-DNA transformation (Hiei, Y., et al., (1994) Plant J. 6:271-82; Hiei,Y., et al., (1997) Plant Molec Bio. 35:205-218; Zhang, J., et al.;(1997) Mol Biotechnol. 8:223-31). Several single copy T-DNA lines(SCTLs) would then be identified by Southern analysis (Ausebel, F. M.,et al., (1987) In: Current protocols in molecular biology, ed. Chanda,V. B. Boston: John Wiley & Sons, Inc.). To test for the efficiency ofT-DNA elimination, a PCR experiment would then be performed onunselected outcross progeny for detecting transmission of the T-DNA.This analysis would be performed on DNA pools from unselected progeny(e.g. a minimum of 384 DNA pools, each pool containing 12 plants). Usingthis evaluation procedure, it is contemplated that other constructs maybe tested, such as those that include a transposase source (explainedbelow), the pollen suicide gene, and the Ds-bar element.

[0250] F. Transposase Expression

[0251] A further embodiment of the instant invention is directed toenriching for independent transpositions while minimizing the recoveryof non-independent ones. One way to accomplish this is by delayingtransposition in development to prevent the early clonal propagation andmeiotic transmission of non-independent events. Control over thedevelopmental timing of transposition is achieved by using heterologouspromoters driving the Ac transposase gene (Rommens, C. M., et al.,(1992) Mol Gen Genet. 231:433-41; Balcells, L. et al., (1994) Plant Mol.Biol. 24:789-98; Scofield, S. R., et al., (1992) Plant Cell. 4:573-82;Swinburne, J., et al., (1992) Plant Cell. 4:583-95; Grevelding, C., etal., (1992) Proc Natl Acad Sci USA. 89:6085-9). Several heterologouspromoters are envisaged in the instant embodiment.

[0252] In a preferred embodiment the strategy to enrich for independenttranspositions is to limit transposase expression exclusively to floraldevelopment, preferably excluding pistil expression (for reasonsexplained below), to prevent vegetative transpositions. Afloral-specific promoter may be used to drive transposase expressionduring the formation of stamen primordia and several are available thatpreclude pistil expression such as rice (Moon, Y. H., et al., (1999)Plant Mol. Biol. 40:167-77; Kang, H. G., et al., (1998) Plant Mol. Biol.38:1021-9; Greco, R., et al., (1997) Mol Gen Genet. 253:615-23) or maizeMADS-box gene promoters (Mena, M., et al., (1996) Science 274:1537-40;Mena, M., et al., (1995) Plant J. 8:845-54). To enhance the frequency oftransposition, both full-length cDNA and a truncated version of Actransposase may be used in rice. The truncated version (ORF103-807) hasbeen shown to enhance the frequency of transposition in heterologousplant species (Houba-Herin, et al., (1990) Mol Gen Genet. 224:17-23; Li,M. G. et al., (1990) Proc Natl Acad Sci USA. 87:6044-8).

[0253] Limiting transposase expression to floral development means thateach floret represents an independent source of transpositions.Statistically, the recovery of non-independent transposition is low—ifpollen from each anther is considered a source of independenttranspositions then the frequency of progeny seed derived from pollenfrom the same anther will be unlikely—at least six times more anthers(six per spikelet) will be produced than seed (one per spikelet).Embodiments of the instant invention include the use of ³⁵S-driventransposase (full-length and truncated) and florally-expressedtransposase constructs transformed into rice along with a simple Dselement inserted into the 5′ UTR of a bar gene. Single copy insert linesare then identified by Southern analysis and these plants are outcrossedto wild type, male-sterile IR36 females. Progeny seedlings are thensubjected to two foliar applications of Finale® (see below) to selectprogeny in which Ds has excised. Based on the mechanism of Ac/Dstransposition (Chen, J., et al., (1987) Genetics. 117:109-116; Chen, J.,et al., (1992) Genetics 130:665-676; Greenblatt, I. M. et al., (1962)Genetics. 47:489-501) more than 50% of these progeny contain linked orunlinked transposed Ds elements. Finale®-resistant progeny from eachstock are analyzed by Southern analysis to determine the effectivenessof recovery of independent transpositions.

[0254] Once transposed, the Ds element needs to be stabilized, yet stillhave the ability to be remobilized. To stabilize transposed Ds elements,the appropriate transposase gene is included within the final T-DNAconstruct containing the Ds launching site. The pollen suicide processor bar antisense strategy eliminates the transposase source along withthe T-DNA, thereby stabilizing any transposed Ds element in progeny.Reintroducing transposase in subsequent generations can easilydestabilize the Ds element, permitting localized mutagenesis ofneighboring genes (Long, D., et al., (1997) Plant J. 11:145-8; Ito, T.,et al., (1999) Plant J. 17:433-44) or reconstitutional (saturation)mutagenesis (Moreno, M. A., et al., (1992) Genetics. 131:939-956; Athma,P., et al., (1992) Genetics. 131; Das, L. et al., (1995) Plant Cell.7:287-94) of any single gene.

EXAMPLES Example 1 Genetic Constructs

[0255] pYU904—Synthetic Ds Element

[0256] The synthetic Ds element was constructed by combining the 5′ and3′ ends of Ac required for transposition. Primers P643(aagctttggccatattgcagtcatcc) (SEQ ID NO:1) and P644(aagcttgctcgagcagggatgaaagtaggatggga) (SEQ ID NO:2) are used to amplifythe 5′ end of the Activator element (Ac) from coordinates 4312 to 4565bp (GenBank Accession X01380) (SEQ ID NO:3) while adding a Hind IIIcloning site to the 3′ end and both a Hind III and Xho I site to the 5′end of the fragment. Primers P645 (gaattccctcgagtagggatgaaaacggtcggtaac)(SEQ ID NO:4) and P646 (gaattcgaatatatgttttcatgtgtgat) (SEQ ID NO:5) areused to amplify the 3′ end of the Ac element from coordinates 1 to 221bp with the additional EcoRI and XhoI restriction sites were added tothe 3′ end of the fragment and an additional EcoRI restriction site wasadded to the 5′ end of the fragment being amplified. These fragmentswere individually cloned in the vector pCR2.1-TOPO (Invitrogen).

[0257] Plasmid pYU890 contained the 5′ end fragment of the Ac element,and plasmid pYU892 contained the 3′ end fragment of the Ac element.

[0258] pYU892 was digested with EcoRI (New England Biolabs), and the 230base pair (bp) Eco RI insert was cloned into the Eco RI site of pUCl9(GenBank Accession M77789) to generate pYU899.

[0259] pYU890 was digested with HindIII (New England Biolabs), and the250 bp insert was subcloned into the HindIII site of plasmid pYU899giving rise to plasmid pYU902. This plasmid contains the 5′ and 3′ endsof Ac, required for transposition, and an internal polylinker site forsubsequent cloning purposes.

[0260] A deletion derivative (pYU903) of pBLUESCRIPT II K/S (Stratagene)was constructed by first digesting with Sac I and Sal I, filling in withKlenow and religated. The plasmid was then digested with Asp 718 and ApaI, filled in with Klenow and relegated. The derivate plasmid representsa deletion of the restriction sites of the KpnI-SacI polylinker but withan intact XhoI cloning site.

[0261] pYU902 was digested with XhoI (New England Biolabs), and theinternal 571 bp fragment was cloned into the XhoI site of pYU903 givingrise to pYU904 (FIG. 4A). This plasmid contains the 5′ and 3′ ends ofAc, and multiple cloning site within the Ds element that are now uniquefor the plasmid. This Ds element is referred to as “Ds-polylinker”.

[0262] pYU905—Ds Element containing Selectable Marker Gene

[0263] A 1.1 kb Sma I fragment containing the bar gene from Streptomyceshygroscopicus (Genbank Accession X17220) (SEQ ID NO:6) is fused to a 0.6kb CaMV ³⁵S promoter fragment (Benfey and Chua, 1990) and 3′polyadenylation signal element (coordinates 514-813) (GenBank AccessionV00090) (SEQ ID NO:7) to create the plasmid pYU117.

[0264] Plasmid pYU 117 was digested with HindI and SnaBI (new EnglandBiolabs) and the 1.8 kb fragment containing the CaMV ³⁵S promoter-bargene-terminator gene was filled in with Klenow fragment DNA polymerase(New England Biolabs). The modified fragment was cloned into the SmaIsite of pYU904 to generate pYU905 (FIG. 4B).

[0265] pYU905 contains the CaMV ³⁵S-driven bar gene inside theDs-polylinker transposable element. This synthetic Ds element is termed“Ds-bar”.

[0266] pYU846—Transposase Source

[0267] The plasmid pKU108A contains a transposase cDNA with a truncatedreading frame (ORFa103-807) (Lee and Starlinger, PNAS 87:6044-6048.1990) was digested with NcoI and BamHI (New England Biolabs). Theinternal 2.1 kb fragment was purified and subcloned into pRTL2(Restrepo-Hartwig and Carrington. J. Virology 66:5662) previouslydigested with the same enzymes. The resulting plasmid, pYU846 (FIG. 5),contained a transcriptional fusion between the CaMV ³⁵S promoter, thetruncated Ac transposase cDNA (amino acids 103-807) and the ³⁵Spolyadenylation sequence.

[0268] GST Constructs

[0269] Two examples of GST constructs based on the barnase gene ofBacillus amyloliquefaciens are shown in Table 1 (SEQ ID NO:15) and Table2 (SEQ ID NO:16). These constructs are derived by replacing thetapetal-specific tobacco promoter TA29 (Genbank Accession A18052) (SEQID NO:8) with a dicot pollen-specific promoter from Arabidopsis thaliana(At59) or monocot pollen-specific promoter from Oryza sativa (rice)(GenBank Accession Z16402) (SEQ ID NO:9).

[0270] The At59 promoter and 5′ UTR is amplified from A. thaliana Col-Ogenomic DNA using primers P755 (acccatgtgagttttctttcttctccat) (SEQ IDNO:10) and P756 (ttataggaaaattccagcagctcagcat) (SEQ ID NO:11). Theseprimers simultaneously amplify the promoter and 5′ UTR sequence whileintroducing a 5′ Pst I cloning site and a 3′ Nco I site situated at thestart of translation. This Nco I site is fused to a 0.74 kb Nco 1-Eco RIsite of the bamase gene containing the nopaline synthase polyadenylationsignal element at the 3′ end to create the At59PSP:barnase:nos transgene(Table 1)(FIG. 3A).

[0271] Likewise, the rice pollen-specific promoter (GenBank AccessionZ16402) (SEQ ID NO:9) is amplified from Orza sativa ssp. indica IR36genomic DNA using primers P731 (gaattccgggccatggcatcctttag) (SEQ IDNO:12) and P732 (ccatggatgatgtggctgcaaatg) (SEQ ID NO:13) which amplifya promoter and 5′ UTR fragment while introducing a 5′ EcoRI site andincluding the 3′ Nco I site at the start of translation. This 0.74 kbEco RI—Nco I fragment is ligated to the Nco I site of the bamase genecontaining the nopaline synthase polyadenylation signal element at the3′ end to create the OsPSP:barnase:Nos transgene (Table 2)(FIG. 3B).

[0272] T-DNA Construct

[0273] The T-DNA vector pPZP200 (GenBank Accession U10460) (SEQ IDNO:14) is digested with Pst I ligated to the 3.2 kb Pst I fragment frompYU846 to generate pYU1001. pYU1001 is digested with Asp 718, filled inwith Klenow, and ligated to the T4 DNA polymerase-treated 1.2 kb Pst1-Eco RI At59:barnase:nos gene fragment (pYU1002) or to the 1.3 kbKlenow-treated Eco RI OsPSP:barnase:nos gene fragment (pYU1003). pYU1002or pYU1003 is digested with Sal I and ligated to the 2.3 kb Xho I Ds-barelement derived from pYU905 to give plasmid pYU1004 containing the GST,transposase source and Ds-bar element. TABLE 1 Sequence and Features ofAT59: Barnase: Nos GST Construct FEATURES Location/Qualifiersmisc_feature 840 . . . 1245 /note = “3′ regulatory sequence containingthe polyadenylation site derived from the nopaline synthase gene fromAgrobacterium T-DNA” 5′ UTR 393 . . . 503 AA changed to CC at 3′ end tocreate NcoI site TATA_signal 364 . . . 368 primer_bind 1 . . . 30 changefrom CCAT to TGCA primer_bind complement (480 . . . 501) primer toamplify At59 promoter and introduce NcoI site CDS 504 . . . 839 /note =“coding sequence of the barnase gene” promoter 1 . . . 392 At59 promoterregion misc_feature 1241 . . . 1246 Eco RI cloning site misc_feature 1 .. . 6 Pst I cloning site BASE COUNT ORIGIN 400 a 232 c 235 g 375 t 1ctgcagggga tttttttaat tacttgtatg ataattattt tcaatagacc tagagacttg (SEQID NO: 15) 61 atatatacta cgtttaataa tcatatgtag tatgtatgat taattaagtaaatacaaaaa 121 tagttacctc aagttttaaa ggtgctattg ggtaattatc tcagtaaaaataatattaga 181 tcaaggcaaa aataactgaa aatatccaga aaagaaggat taaacaaaggcatccaaaat 241 ctataattgg gttttttgga gaaatgacca tagagattta aatcaatggttgtctaatct 301 atgttaattc tcaatcctct attgactctt ctcatctcct tttctctctccccagttcct 361 ggttattaaa gcaatcaggt gattcaaatc tttaatcttt taatcccggcaggcctatct 421 gaaacaacaa cctccgtttg aggttttgcc gggaaaatat aaagttcacaggctttggtc 481 tctgcatttg caatatattt accatggtac cggttatcaa cacgtttgacggggttgcgg 541 attatcttca gacatatcat aagctacctg ataattacat tacaaaatcagaagcacaag 601 ccctcggctg ggtggcatca aaagggaacc ttgcagacgt cgctccggggaaaagcatcg 661 gcggagacat cttctcaaac agggaaggca aactcccggg caaaagcggacgaacatggc 721 gtgaagcgga tattaactat acatcaggct tcagaaattc agaccggattctttactcaa 781 gcgactggct gatttacaaa acaacggacc attatcagac ctttacaaaaatcagataac 841 gaaaaaaacg gcttcctgcg gaggccgttt ttttcagctt tacataaagtgtgtaataaa 901 tttttcttca aactctgatc ggtcaatttc actttccggn nnnctctagaggatccgaag 961 cagatcgttc aaacatttgg caataaagtt tcttaagatt gaatcctgttgccggtcttg 1021 cgatgattat catataattt ctgttgaatt acgttaagca tgtaataattaacatgtaat 1081 gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaattatacatttaat 1141 acgcgataga aaacaaaata tagcgcgcaa actaggataa attatcgcgcgcggtgtcat 1201 ctatgttact agatcgggaa gatccccggg taccgagctc gaattc

[0274] TABLE 2 Sequence and Features of OsPSP: Barnase: Nos GSTConstruct FEATURES Location/Qualifiers misc_feature 1208 . . . 1213/note = “Eco RI cloning site” misc_feature 807 . . . 1212 /note = “3′regulatory sequence containing the polyadenylation site derived from thenopaline synthase gene from Agrobacterium T-DNA” CDS 471 . . . 806 /note= “coding sequence of the barnase gene” misc_feature 1 . . . 6 /note =“Eco RI cloning site” promoter 6 . . . 470 /note = “OsPSP promoterregion” BASE COUNT ORIGIN 374 a 279 c 239 g 317 t 1 gaattccgggccatggcatc ctttagaatg gaggaattta agtgaaattg agctaaacta (SEQ ID NO: 16)61 tgtgaacatc ctatgaagtt actgcattca aggcgcccaa catgaaatct attcaggttc 121ccaagttgtg ggcttccgta acgtcaaaat tcgacagatt tctggctggc taaaacaccc 181acaacggcaa taatagcctc gctcgtcaaa acattcaccc atttttagct tggtcatcat 241caaaagtagg atcaaatcaa caatctgcct tctcttcagc cactcgatcc caacggcatc 301tccaacgatt cctacttgaa ggacagccat ggaaatcctc caggttcccc aggttactta 361taccacagct cgaatccgtt ccaaaccagg ccatttcagt accctcctct cacattttcc 421ccaaataata atagaggaag gggaaaaaca catttgcagc cacatcatcc atggtaccgg 481ttatcaacac gtttgacggg gttgcggatt atcttcagac atatcataag ctacctgata 541attacattac aaaatcagaa gcacaagccc tcggctgggt ggcatcaaaa gggaaccttg 601cagacgtcgc tccggggaaa agcatcggcg gagacatctt ctcaaacagg gaaggcaaac 661tcccgggcaa aagcggacga acatggcgtg aagcggatat taactataca tcaggcttca 721gaaattcaga ccggattctt tactcaagcg actggctgat ttacaaaaca acggaccatt 781atcagacctt tacaaaaatc agataacgaa aaaaacggct tcctgcggag gccgtttttt 841tcagctttac ataaagtgtg taataaattt ttcttcaaac tctgatcggt caatttcact 901ttccggnnnn ctctagagga tccgaagcag atcgttcaaa catttggcaa taaagtttct 961taagattgaa tcctgttgcc ggtcttgcga tgattatcat ataatttctg ttgaattacg 1021ttaagcatgt aataattaac atgtaatgca tgacgttatt tatgagatgg gtttttatga 1081ttagagtccc gcaattatac atttaatacg cgatagaaaa caaaatatag cgcgcaaact 1141aggataaatt atcgcgcgcg gtgtcatcta tgttactaga tcgggaagat ccccgggtac 1201cgagctcgaa ttc

Example 2 Preventing or Eliminating Transmission of a Transgene

[0275] When hemizygous, eliminating transmission of a transgene locus isachieved by linking a gene of interest to a suicide gene under thecontrol of a male- or female-specific promoter. This construct, termedthe “gametophytic suicide trait” (GST) induces cell death that isrestricted to the microspores or megaspores which receive the GST,thereby effectively reducing or eliminating transmission of the gene ofinterest that is linked to the GST.

[0276] The gene carrying the special trait (i.e., transgene of interest)that you want to eliminate from transgenic pollen can be placed anywhereas long as it is in physical proximity to the GST. Because the GSTtransgene complex is hemizygous, there will be complete linkage in theGST transgene complex and there is no concern that the GST and othergenes and/or transgenes will recombine.

[0277] The methods of this invention can be used with in planta or seedtransformation techniques that do not require culture or regeneration.Examples of these techniques are described in Bechtold, N., et al.(1993) CR Acad. Sci. Paris/Life Sciences 316:1118-93; Chang, S. S., etal. (1990) Abstracts of the Fourth International Conference onArabidopsis Research, Vienna, p. 28; Feldmann, K. A. and Marks, D. M(1987) Mol. Gen. Genet. 208:1-9; Ledoux, L., et al. (1985) ArabidopsisInf Serv. 22:1-11; Feldmann, K. A. (1992) In: Methods in ArabidopsisResearch (Eds. Koncz, C., Chua, N-H, Schell, J.) pp. 274-289; Chee, etal., U.S. Pat. Ser. No. 5,376,543.

[0278] Arabidopsis.

[0279] Plasmids containing the GST constructs (i.e., At59PSP:barnase:nosor OsPSP:barnase:Nos) linked to the bar transgene are transformed byelectroporation into Agrobacterium and then into Arabidopsis using thevacuum infiltration method (Bechtold et al., 1993, supra). As discussedpreviously, the bar gene construct codes for phosphinothricin acetyltransferase (PAT) driven by the CaMV ³⁵S promoter to provide resistanceto phophinothricin (PPT).

[0280] Transformants are selected based on resistance to PPT, and T2seed is generated from a number of independent lines. This seed isplated on GM media containing various concentrations of herbicide andscored for germination and survival. Multiple transgenic linesoverexpressing either the wild type or the resistant mutant producesignificant numbers of green seedlings on an herbicide concentrationthat is lethal to the empty vector control.

[0281] The transgene or gene of interest to be included in the transgenecomplex and the initial transgenic lines need only be characterized fornumber of transgene loci and complexity of the transgene insertion inorder to identify lines with single-copy, non-tandemly duplicatedinsertions. Characterization of the initial transgenics is accomplishedby PCR and/or Southern analysis, both methods being well known to thoseskilled in the art of DNA amplification and gel electrophoresis.

[0282] Transformed plants hemizygous for the GST/transgene of interest,in this example the bar gene, are grown under the same growingconditions with transformed plants homozygous/heterozygous for thetransgene of interest alone (i.e., no GST is present with the transgene)and with control, wild-type plants (i.e., plants lacking both GST andthe transgene of interest) using appropriate statistical procedures(e.g., randomized complete block design or lattice design). Pollen iscollected from the each of the individual plants and analyzed for thetransgene and/or controlled crosses to wild-type plants are conductedand the seed is collected on a per plant basis and the resultantseeds/plants are analyzed for the transgene.

[0283] Plants hemizygous for the GST/transgene complex and wild-typeplants produce pollen and/or seeds/plants all of which fail to containthe transgene of interest. In contrast, plants heterozygous for thetransgene produce pollen and seeds/plants which show normal segregationfor the transgene of interest. Plants homozygous for the transgene ofinterest produce pollen all of which contain the transgene of interest.Plants homozygous for the transgene of interest when crossed towild-type plants produce F2 seeds/plants which show normal segregationpatterns for the transgene of interest. Thus, plants hemizygous for theGST/transgene complex fail to produce pollen with the transgene whileplants homozygous or heterozygous for the transgene alone produce atleast some pollen which does contain the transgene of interest.

[0284] This is the same situation for the dispersed transposition aspectof the invention. The initial transgene complex carries both the GST andtransposon and/or transposase. After the transgenic lines are generated,they are selected for dispersed transpositions. That is, one or twotransgenic lines are all that are needed for subsequent selections. Nofurther transformation is necessary. An additional, optional transgene,such as the CaMV ³⁵S promoter/bar gene construct can be added if desiredto aid in the selection of transformants.

[0285] Turfgrass

[0286] A nucleic acid construct (GST) in which the gene encoding bamaseunder the control of a pollen-specific promoter from maize is made suchthat the Roundup® resistance gene is linked to the GST. A virgin (i.e.,wild-type) turfgrass genome is transformed with the transgene complexcontaining the three elements (maize pollen-specific promoter, barnasegene, Roundup® resistance gene) such that the resultant transgenic plantis hemizygous for the transgene complex.

[0287] The transgenic plant is vegetatively propagated to yield progenyplants that are also hemizygous for the transgene complex. Although allplants generated asexually from the transgenic plants are resistant toRoundup® treatment, transmission of the Roundup® resistance gene viacross-pollination is eliminated because no viable transgenic pollen isproduced.

[0288] Since the GST construct is male-specific, the hemizygotictransgenic lines can be maintained by crossing to wild-type pollen. Whentransgene elimination is required (e.g., in selection for dispersedtranspositions) then the hemizygotic transgenic lines are used as males(pollen donors) and crossed to wild-type females. In this instance, onlynon-transgenic pollen or pollen containing dispersed transpositionswould be propagated. For the purposes of eliminating unwanted pollentransmission of a transgene (e.g., herbicide resistance in turfgrass),the hemizygotic transgenic lines can be planted and only wild-typepollen will survive.

[0289] Alfalfa

[0290] A nucleic acid construct (GST) in which the gene encodingbarnase, under the control of a pollen-specific promoter from rice, ismade such that a Bt gene is linked to the GST. A virgin (i.e.,wild-type) alfalfa genome is transformed with the transgene complexcontaining the three elements (rice pollen-specific promoter, barnasegene, Bt gene) such that the resultant transgenic plant is hemizygousfor the transgene complex.

[0291] The hemizygotic transgenic plant is vegetatively propagated toyield progeny plants that are also hemizygous for the transgene complex.Although the plant is resistant/tolerant to certain lepidopteran insectpests, transmission of the Bt gene via cross pollination is eliminatedbecause no viable transgenic pollen is produced.

[0292] Transformed Corn

[0293] In the case of corn, the GST/transgene complex is inserted intothe corn genome and the “female” parent carries the transgene complex.Upon hybridization with wild-type pollen, only 1/2 of the progeny hybridseed will carry the transgene complex (not a problem for functionalgenomics applications). This limitation is circumvented by using a flpor lox recombinase system—the GST trait is kept inactive and homozygousuntil the hybrid is produced. At that point, frt- or cre-mediatedrecombination activates the GST trait (e.g., by removing a DNA block totranscription or by activating transcription), now present in all hybridprogeny instead of 1/2. The transgene complex containing activated GSTis eliminated from any pollen inheriting the transgene complex (e.g. 50%of the meiotic products).

Example 3 Enriching Dispersed Transposition Events

[0294] By physically linking the sterility trait to a transposonlaunching site and/or transposase source, the “genetic sterility filter”is used to highly enrich for dispersed and/or stabilized transpositionevents without the use of chemicals and without the need to selectagainst progeny containing linked transposition events and/ortransposase source.

[0295] For instance, when 50% pollen sterility is achieved, theremaining viable haploid genomes will not have inherited the suicidegene and its associated elements such as the transposon launching siteand/or transposase gene because of normal homologous chromosomesegregation, independent assortment and meiotic recombination. Afraction of these viable genomes will contain newly transposed elements,especially those elements that have assorted independently or recombinedfrom the launching site and its associated suicide gene. Therefore, the“genetic sterility filter” eliminates gametes containing transposedelements that remain linked to the launching site and/or gametescontaining a transposase gene.

[0296] If the remaining viable pollen is used to fertilize ovules,either by controlled pollinations or by wind-pollination, a fraction ofthe resultant progeny will contain transposed elements. These progenyare readily identified by the inclusion of a selectable or screenablemarker inside of the transposon, such as the petunia, Arabidopsis, orAgrobacterium CP4 EPSPS gene (Padgette, S. R. et al., (1987) ArchBiochem Biophys Vol. 258/2:564-73; Klee, H. J. et al., 1987 Mol GenGenet Vol. 210/3:437-42; Hoef, A., et al., (1998) Food Addit ContamVol.15/7:767-74; Harrison, L. A., et al., (1996) J Nutr Vol.126/3:728-40), encoding glyphosate (Roundup@) resistance (Malik, J., etal., (1989) Biofactors Vol. 2/1:17-25); or a variety of acetolactatesynthase (ALS) genes, encoding resistance to sulfonylurea herbicides(Whitcomb, C. E. (1999) Toxicol Ind Health Vol. 15/1-2:231-9), such asthe Arabidopsis multiherbicide-resistant gene, csrl-4 (Mourad, G. etal., (1994) Mol Gen Genet Vol.: 243/2:178-84), or the bar gene fromStreptomyces (Thompson, C. J et al., (1986) EMBO J. Vol. 6:2519-2523),encoding resistance to phosphinothricin (Finale®), to name a few.

[0297] In the case of the transmission of an autonomous element, such asAc, progeny containing dispersed transposed Ac elements are identifiedby classical genetic means such as transactivation of a Ds-inducedreporter gene. In one embodiment of the instant invention, thetransposon is constructed with a pollen survival gene that permits onlyviable pollen that contain transposed elements, thereby completelyeliminating the need for chemical selection or screens.

[0298] If the GST complex additionally contains a transposable element,then the frequency of transposition (both to linked and unlinked sites)can be high, depending on the source of transposase and other factors.Assuming a transposition frequency of 5%, 70% of which may be linked,then 30% are unlinked and 15% of these (random independent assortment ofthe transposon with the GST chromosome) will be recovered in progeny andeasily identified by herbicide resistance contained on the transposableelement. If we want to recover 100,000 independent insertions, anestimate of the number of seed required would be:(100,000×2/0.3)/0.05=13,333,333 F1 seed needed. These could be generatedand screened (recovered as individual plants) in less than 3 years.

Example 4 Enriching Stably Dispersed Transposition Events

[0299] A further embodiment of the instant invention is directed toinclusion, in the transgene complex, in addition to the transposonlaunching site and the suicide gene, other genes such as a transposasesource. In this embodiment, the “genetic sterility filter” enriches fordispersed elements while also eliminating the transmission of thetransposase source to progeny.

[0300] The simultaneous elimination of the transposon donor site andtransposase gene has the added benefit of transmitting transposedelements that are stabilized (i.e. no longer transposing due to loss ofthe transposase gene) thereby preventing additional transpositions(secondary transpositions) from occurring.

[0301] Other embodiments of the instant invention include positioningthe transposase source and transposon launching sites in separatetransgene complexes. For instance, the launching site and thetransposase source can be brought together in one genome on separateelements to achieve the same enrichment for dispersed transpositions.Moreover, in cases where localized transposition is desirable, i.e., tosaturate a specific chromosomal region with insertions or to recoverinsertions in nearby genes of interest, the transposase source iseliminated by the sterility filter method without necessarilyeliminating linked transpositions.

Example 5 Rice—A Model Plant System

[0302] A. Rice Transformation

[0303] Rice transgenics are generated employing a 24-wellmicrotiter-based method that permits high throughput transgenicproduction. The method is an adaptation of published protocols (Hiei,Y., et al., (1994) Plant J. 6:271-82; Hiei, Y. et al. (1997) Plant MolecBio. 0.35:205-218; Zhang, J., et al., (1997) Mol Biotechnol. 8:223-31)and involves an entirely liquid culturing and transformation system thatallows the production of transgenic from scutellar callus induction,co-cultivation with Agrobacterium, treatment with Timentin, and shootregeneration. This system is used to generate ca. 50-100 independenttransgenic lines each month.

[0304] A tissue sample from each transgenic line is collected, DNAextracted and analyzed by Southern to identify lines that contain singlecopy T-DNA inserts (SCTLs). Shoots from SCTLs are micropropagated,rooted and transplanted to soil according to methods well known to oneskilled in the art.

[0305] B. Genetic Methodologies

[0306] A broad-based, high volume-crossing program is available at CIATfor the generation of stocks, seed and transposon lines. Typically,about 1000 controlled (hand) crosses are made each cycle, includingsingle, doubled, top and backcrosses. Number of F1 seeds obtaineddepends on the cross type and breeding objectives.

[0307] Crosses can be made throughout the year under biosafety-approvedscreenhouse conditions. Three plantings of parents at intervals of from7 to 10 days are made to assure simultaneous flowering. Parents aregrown in large pots or grown in the field in a hybridization block whenemploying the male-sterile female line.

[0308] For hand pollinations, the methods for crossing are: selection ofparent plants, emasculation of panicles (removal of anthers for thefemale parent), covering of emasculated florets with a glassine bag,pollination of female parent with pollen collected from the male parent,covering of the pollinated panicle with the glassine bag, andidentification of panicles used for crossing with a crossing tagcontaining relevant information about the parents, dates, and name ofthe person who did the crossing. For detailed information on thehybridization of rice see, for example, Coffman, W. R. and R. M. Herrera(1980) Rice, In: Hybridization of Crop Plants, W. R. Fehr and H. H.Hadley, Editors, Chapter 36: 511-522, American Society of Agronomists.

[0309] Selected F1 and T1 plants are harvested individually to produceF2 seeds. About 25 days after pollination hybrid seed is harvested,threshed, cleaned, placed in coin envelopes. This seed is stored underlow humidity and temperature. T1 seed are grown in flats and selectedfor herbicide resistance by two foliar applications of 0.05% Finale® at25 and 35 days after germination.

[0310] C. Sample Tracking

[0311] A relational, barcode sample tracking database can be used totrack plants, seed, and DNA samples through the workflow. Eachtransgenic that is created is assigned a unique (alphanumeric)identifier. Stock plants derived from transgenics carry this identifierand a second unique stock identifier. All stocks that enter theproduction nurseries carry the transgenic/stock identifiers andtestcross seed derived from each stock are assigned a unique identifierfor each cross (T1 seed lot).

[0312] After Finale® selection, each resistant plant is assigned aunique line identifier. This information is coded on a plant label withtwo tearoff labels; each one contains identical alphanumericalidentifiers and associated barcode. One tearoff label is attached totissue sample and the other remains on the plant, ultimately stapled tothe seed package. A widget bar code reader logs tissue samples into adatabase that then tracks samples through the DNA extraction queue andultimately into a position of a 384-microtiter plate used for PCRamplification and DNA sequencing.

[0313] D. High-Throughput DNA Isolation and Normalization

[0314] Tissue samples are lyophilized-prior to DNA extraction.Lyophilized tissue for shipping in plastic bags is sealed withdesiccant. Tissue is ground with zirconium silica beads (ATGC, Inc.) byrapidly shaking 96-384 tubes on a commercial 10 gal paint shaker (FluidDynamics, Inc.).

[0315] The DNA is extracted using a high throughput, parallel methodadapted from previously published methods (Galbiati, M., et al., (2000)Functional & Integrative Genomics, in press) and the purified DNA isstored in 96-format microtiter plate. DNA concentration is measuredrobotically by the Picogreen method (Molecular Probes, Inc.) using aSpectrofluor plate reader and Genesis robotics workstation (Tecan,Inc.).

[0316] To make a second working plate for PCR analysis, DNA isnormalized robotically by removing a fixed amount of DNA from each welltogether with the appropriate amount of buffer into a second microtiterplate. Mother-daughter replicas of these normalized working plates aremade robotically and samples are shipped for amplification andsequencing.

[0317] The feasibility of this entire procedure, including scripts, fromtissue extraction to normalization, has been enabled for both maize andArabidopsis.

[0318] E. Selection of Transposon Lines Selection of transposon linesinvolves transgenic production and genetic testing for the recovery ofsingle copy lines (SCLs), scaleup, sample tracking, processing andshipping followed by a production phase. Methods including DNAfingerprinting of T-DNA both in Agrobacterium inoculants and in primaryrice transgenics, Agrobacterium-mediated transformation protocols, andmethods for rice regeneration, micropropagation, and transplantation tosoil may be employed.

[0319] Tissue is collected from each transgenic line, DNA extracted andanalyzed by Southern analysis to identify single copy T-DNA lines(SCLs). All SCLs may be maintained in shoot culture and shipped formicropropagation. Rooted plantlets may be transplanted to soil andtested for transposition rates.

[0320] F. Stock Production

[0321] Genetic data on transposition frequencies is used to select forthe best transgenic lines for continual stock production. Rooted shootsare transplanted to soil and further propagated by simple division toincrease the numbers of male plants in the production nursery.

[0322] In one embodiment, if transpositions are limited to stamendevelopment, an alternative, but not mutually exclusive method of stockpropagation, is to cross wild-type pollen to the female transgenicplants—T-DNA elimination and transposition will not occur in thisdirection. Therefore, this seed can simply be replanted each season togenerate additional stock for new transposition selections.

[0323] The flowering time for the rice variety Nipponbare is 60 dayswhen grown at or near the equator. By using an equatorial location it ispossible to achieve nearly 4 plant generations each year. Theabbreviated generation time and constant year-round growing conditionsin Cali, Mexico make rice genetics nearly as efficient as Arabidopsis.

[0324] G. Foundation Seed Production

[0325] In one embodiment of the instant invention, the objective is toselect for single, dispersed transpositions and to recover theseinsertions in hemizygous condition. This permits the recovery ofnon-lethal, recessive lethal and female haplo-insufficient mutations.

[0326] To produce T1 seed, stock plants are crossed as males tomale-sterile female plants in the nursery. Efficient outcrossing in ricecan be achieved by interplanting males and male-sterile females.Male-sterility in Nipponbare is not presently available so a nuclearmale sterility (ms) mutation is introgressed from an O. sativa ssp.indica strain into Nipponbare in the nurseries. The introgressionprocess is greatly accelerated by marker-assisted mapping and breeding,selecting backcross progeny for the ms mutation and Nipponbare markers.

[0327] The ms locus is first mapped using previously characterizedsimple sequence repeat (SSR) markers (McCouch, S. R., et al., (1997)Plant Mol. Biol. 35:89-99; Panaud, O., et al., (1996) Mol Gen Genet.252:597-607). 7000 SSR sequences are available from the Monsanto roughdraft (www.rice-research.org). Linked SSRs are used to follow the msallele during introgression, while unlinked SSRs throughout the genomeare used to select against donor germplasm.

[0328] Two backcrosses and one self pollination should be sufficient totransfer the ms allele to the Nipponbare background. The F1 crosses aremade and F2 progeny are generated. The introgressed ms line is madeavailable for T1 seed production. A 1:1 line segregating for the msphenotype is used in the nursery, genotyped by SSR analysis, and themale-fertile plants culled. Transposon stocks are hybridized to ms/msfemale plants to generate T1 seed for Ds line selection.

[0329] H. Transposon Line Selection

[0330] T1 seed is planted in flats and 25-day-old seedlings are selectedby a foliar application of 0.05% Finale® followed by a second treatment10-15 days later. Finale®-resistant plants are bar-coded andtransplanted to the nursery and allowed to self-pollinate to generate T2seed.

[0331] A tissue sample (ca. 1-2 gm leaf tissue) from each plant iscollected, placed in bar-coded tubes, lyophilized and prepared for DNAextraction.

[0332] T2 seed from each line are collected, threshed, placed inbar-coded envelopes and prepared for storage or public distribution.

[0333] While the invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1 16 1 26 DNA Artificial Sequence Description of Artificial Sequence 5′PCR primer 1 aagctttggc catattgcag tcatcc 26 2 35 DNA ArtificialSequence Description of Artificial Sequence 5′ PCR primer 2 aagcttgctcgagcagggat gaaagtagga tggga 35 3 4565 DNA Zea mays Transposable elementAc 3 tagggatgaa aacggtcggt aacggtcggt aaaatacctc taccgttttc attttcatat60 ttaacttgcg ggacggaaac gaaaacggga tataccggta acgaaaacga acgggataaa 120tacggtaatc gaaaaccgat acgatccggt cgggttaaag tcgaaatcgg acgggaaccg 180gtatttttgt tcggtaaaat cacacatgaa aacatatatt caaaacttaa aaacaaatat 240aaaaaattgt aaacacaagt cttaattaaa catagataaa atccatataa atctggagca 300cacatagttt aatgtagcac ataagtgata agtcttgggc tcttggctaa cataagaagc 360catataagtc tactagcaca catgacacaa tataaagttt aaaacacata ttcataatca 420cttgctcaca tctggatcac ttagcatgca taaactatta caaccaaggc tcatctgtca 480acaaacataa gacacattgc tcatggagag gagccacttg ctacatcttc attattctta 540gaaaattcta ttgcgtcttc atcctgttaa tacacaaaaa taagtcagtt ttggataaat 600aaatacatat agaagaacat gaattgatat gcagggagta taaataaata catataggag 660aacatgaatc tgtgaactaa cacggctggg agctaggcag ctagcagcta gcgcctaaca 720gctgggagcc taacagctag cagctagcag ccaatcaaaa caaggcgaca aggcgcatgc 780agtgagatca aaaatctgtt aatgccagcc atgcagggag tataacacgg ctgggcagca 840aggcgcatgc atcaaaacaa ggcgacagca aacagcccat gcatcaaaac agtagtgaat 900aatagcaaat taatagccca tgcacgaagt aaataataat ctttaaatac ctcatccata 960tgattctcat gatttgttgc agcagcaata acagagtcta gcacctcgag atcaccaatc 1020attgttggaa aatatgtagc accttgaatg acacaaatat gcatcaatat aagtaaaata 1080attgttgaat aactataaat tggaacttca ttataacata tatgcattca ccttttctag 1140atgctgctac ccaatctttt gtgcatatca aagcttcaac aatctccgaa ccaagacgat 1200tgcggtaagg atcaacaaca cgaccaccag cactgaacgc agactcagaa gcaacagttg 1260acacttgtat tgctagcaca tcccttgcaa tttgggtgag aataggatat tctgcaaccc 1320ttcccctcca ccatgataaa atatcaaact gaccactatg cttcaaaagg ggttcagaca 1380tatatttatc caattcattt gactctactt gatcataatc cttcaactca tgcaaatagt 1440tttgaaattc atcatcttca ttttccatca aggtatcatc catactatca ttagtagttg 1500tctttgtctt tggagctgaa ggactacaac tagaatagaa ttgatacaat tttctaatga 1560ccctaacaaa gtcatctaca tgaactttgt atgaatcacc atgaaatttt ttcatataga 1620actcaatcaa tattttcttg tacctagggt caaggaagca tgctacagct agtgcaatat 1680tagacacttt ccaatatttc tcaaactttt cactcattgc aacggccatt ctcctaatga 1740caaatttttc atgaacacac cattggtcaa tcaaatcctt tatctcacag aaacctttgt 1800aaaataaatt tgcagtggaa tattgagtac cagataggag ttcagtgaga tcaaaaaact 1860tcttcaaaca cttaaaaaga gttaatgcca tcttccactc ctcggcttta ggacaaattg 1920catcgtacct acaataattg acatttgatt aattgagaat ttataatgat gacatgtaca 1980acaattgaga caaacatacc tgcgaggatc acttgtttta agccttatta gtgcaggctt 2040ataatataag gcatccctca acatcaaata ggttgaattc catctagttg agacatcata 2100tgagatccct ttagatttat ccaagtcaca ttcactagca cacttcatta gttcttccca 2160ctgcaaagga gaagatttta cagcaagaac aatcgctttg attttctcaa ttgttcctgc 2220aattacagcc aagccatcct ttgcaaccaa gttcagtatg tgacaagcac acctcacatg 2280aaagaaagca ccatcacaaa ctagatttga atcagtgtcc tgcaaatcct caattatatc 2340gtgcacagct acttcatttg cactagcatt atccaaagac aaggcaaaca attttttctc 2400aatgttccac ttaaccatga ttgcagtgaa ggtttgtgat aacctttggc cagtgtggcg 2460cccttcaaca tgaaaaaagc caacaattct tttttggaga caccaatcat catcaatcca 2520atggatggtg acacacatgt atgacttatt ttgacaagat gtccacatat ccatagttgt 2580actgaagcga gactgaacat cttttagttt tccatacaac ttttcttttt cttccaaata 2640caaatccatg atatattttc tagcagtgac acgggacttt attggaaagt gagggcgcag 2700agacttaaca aactcaacaa agtactcatg ttctacaata ttgaaaggat attcatgcat 2760gattattgcc aaatgaagct tctttaggct aaccacttca tcgtacttat aaggctcaat 2820gagatttatg tctttgccat gatccttttc actttttaga cacaactgac ctttaactaa 2880actatgtgat gttctcaagt gatttcgaaa tccgcttgtt ccatgatgac cctcagccct 2940atacttagcc ttgcaattag gaaagttgca atgtccccat acctgaacgt atttctttcc 3000atcgacctcc acttcaattt ccttcttggt gaaatgctgc catacatccg atgtgcactt 3060ctttgccctc ttctgtggtg cttcttcttc gggttcaggt tgtggctgtg gttgtggttc 3120tggttgtggt tgtggttgtg gttgtggttc atgaacaata gccatatcat cttgactcgg 3180atctgtagct gtaccatttg cattactact gcttacactc tgaataaaat gcctctcggc 3240ctcagctgtt gatgatgatg gtgatgtgcg gccacatcca tgcccacgcg cacgtgcacg 3300tacattctga atccgactag aagaggcttc agcttttctt ttcaaccctg ttataaacag 3360atttttcgta ttattctaca gtcaatatga tgcttcccaa tctacaacca attagtaatg 3420ctaatgctat tgctactgtt tttctaatat ataccttgag catatgcaga gaatacggaa 3480tttgttttgc gagtagaagg cgctcttgtg gtagacatca acttggccaa tcttatggct 3540gagcctgagg gaggattatt tccaaccgga ggcgtcatct gaggaatgga gtcgtagccg 3600gctagccgaa gtggagagca gagccctgga cagcaggtgt tcagcaatca gcttggtgct 3660gtactgctgt gacttgtgag cacctggacg gctggacagc aatcagcagg tgttgcagag 3720cccctggaca gcacacaaat gacacaacag cttggtgcaa tggtgctgac gtgctgtact 3780gctaagtgct gtgagcctgt gagcagccgt ggagacaggg agaccgcgga tggccggatg 3840ggcgagcgcc gagcagtgga ggtctggagg accgctgacc gcagatggcg gatggcggat 3900gggcggaccg cggatgggcg agcagtggag tggaggtctg ggcggatggg cggaccgcgg 3960cgcggatggg cgagtcgcga gcagtggagt ggagggcgga ccgtggatgg cggcgtctgc 4020gtccggcgtg ccgcgtcacg gccgtcaccg cgtgtggtgc ctggtgcagc ccagcggccg 4080gccggctggg agacagggag agtcggagag agcaggcgag agcgagacgc gtcgccggcg 4140tcggcgtgcg gctggcggcg tccggactcc ggcgtgggcg cgtggcggcg tgtgaatgtg 4200tgatgctgtt actcgtgtgg tgcctggccg cctgggagag aggcagagca gcgttcgcta 4260ggtatttctt acatgggctg ggcctcagtg gttatggatg ggagttggag ctggccatat 4320tgcagtcatc ccgaattaga aaatacggta acgaaacggg atcatcccga ttaaaaacgg 4380gatcccggtg aaacggtcgg gaaactagct ctaccgtttc cgtttccgtt taccgttttg 4440tatatcccgt ttccgttccg ttttcgtttt ttacctcggg ttcgaaatcg atcgggataa 4500aactaacaaa atcggttata cgataacggt cggtacggga ttttcccatc ctactttcat 4560ccctg 4565 4 36 DNA Artificial Sequence Description of ArtificialSequence 3′ PCR primer 4 gaattccctc gagtagggat gaaaacggtc ggtaac 36 5 29DNA Artificial Sequence Description of Artificial Sequence 3′ PCR primer5 gaattcgaat atatgttttc atgtgtgat 29 6 615 DNA Streptomyceshygroscopicus bar gene for phosphinothricin acetyl transferase 6gaattcgagc tcggtacccg gggatctacc atgagcccag aacgacgccc ggccgacatc 60cgccgtgcca ccgaggcgga catgccggcg gtctgcacca tcgtcaacca ctacatcgag 120acaagcacgg tcaacttccg taccgagccg caggaaccgc aggagtggac ggacgacctc 180gtccgtctgc gggagcgcta tccctggctc gtcgccgagg tggacggcga ggtcgccggc 240atcgcctacg cgggcccctg gaaggcacgc aacgcctacg actggacggc cgagtcgacc 300gtgtacgtct ccccccgcca ccagcggacg ggactgggct ccacgctcta cacccacctg 360ctgaagtccc tggaggcaca gggcttcaag agcgtggtcg ctgtcatcgg gctgcccaac 420gacccgagcg tgcgcatgca cgaggcgctc ggatatgccc cccgcggcat gctgcgggcg 480gccggcttca agcacgggaa ctggcatgac gtgggtttct ggcagctgga cttcagcctg 540ccggtaccgc cccgtccggt cctgcccgtc accgagatct gatgacccgg gggatccctg 600caggcatgca agctt 615 7 831 DNA Agrobacterium tumefaciens polyA_signal(514)..(813) 7 cgagcatttt atggattttc ttcagatgag actagttcaa gcttgaaaattaagcccccc 60 ccccgaaatc atcgccagag gtcgtcccag cccggcatct atatatagcgccaatatagt 120 ttgtcttaca caaacacacc tcacatcatg aatttcgcag atactcccttggcctccctc 180 gacctagact gggcatgcga agagtttatc aaaacttatg gtgcatctccacaattggaa 240 acaggagagg taatccaaac aaacaatggg ctgctgtatt tgtatggcaaaggttcactc 300 tcacagcgga ttcatgacac acacctcaaa tttaaggaga aggaagaattatccttcact 360 accataaagc cagctgagat gaaggcgcaa caaagtgatt taacttattatgtcgccatt 420 tttcaaagca actatttcct gtgcgtttca aatccagaga aaggctttctgagatgccat 480 aatcgcccat ttctgtaccc catagtagcc catggatcga tgagctaagctagctatatc 540 atcaatttat gtattacaca taatatcgca ctcagtcttt catctacggcaatgtaccag 600 ctgatataat cagttattga aatatttctg aatttaaact tgcatcaataaatttatgtt 660 tttgcttgga ctataatacc tgacttgtta ttttatcaat aaatatttaaactatatttc 720 tttcaagata tcattcttta caagtatacg tgtttaaatt gaataccataaatttttatt 780 tttcaaatac atgtaaaatt atgaaatggg agtggtggcg accgagctca a831 8 1287 DNA Artificial Sequence Description of Artificial SequenceConstruct- TA29barnaseA. tumefaciens poly-A site 8 atctagctaa gtataactggataatttgca ttaacagatt gaatatagtg ccaaacaaga 60 agggacaatt gacttgtcactttatgaaag atgattcaaa catgattttt tatgtactaa 120 tatatacatc ctactcgaattaaagcgaca taggctcgaa gtatgcacat ttagcaatgt 180 aaattaaatc agtttttgaatcaagctaaa agcagacttg cataaggtgg gtggctggac 240 tagaataaac atcttctctagcacagcttc ataatgtaat ttccataact gaaatcaggg 300 tgagacaaaa ttttggtactttttcctcac actaagtcca tgtttgcaac aaattaatac 360 atgaaacctt aatgttaccctcagattagc ctgctactcc ccattttcct cgaaatgctc 420 caacaaaagt tagttttgcaagttgttgtg tatgtcttgt gctctatata tgcccttgtg 480 gtgcaagtgt aacagtacaacatcatcact caaatcaaag tttttactta aagaaattag 540 ctaccatggt accggttatcaacacgtttg acggggttgc ggattatctt cagacatatc 600 ataagctacc tgataattacattacaaaat cagaagcaca agccctcggc tgggtggcat 660 caaaagggaa ccttgcagacgtcgctccgg ggaaaagcat cggcggagac atcttctcaa 720 acagggaagg caaactcccgggcaaaagcg gacgaacatg gcgtgaagcg gatattaact 780 atacatcagg cttcagaaattcagaccgga ttctttactc aagcgactgg ctgatttaca 840 aaacaacgga ccattatcagacctttacaa aaatcagata acgaaaaaaa cggcttcctg 900 cggaggccgt ttttttcagctttacataaa gtgtgtaata aatttttctt caaactctga 960 tcggtcaatt tcactttccggnnnnctcta gaggatccga agcagatcgt tcaaacattt 1020 ggcaataaag tttcttaagattgaatcctg ttgccggtct tgcgatgatt atcatataat 1080 ttctgttgaa ttacgttaagcatgtaataa ttaacatgta atgcatgacg ttatttatga 1140 gatgggtttt tatgattagagtcccgcaat tatacattta atacgcgata gaaaacaaaa 1200 tatagcgcgc aaactaggataaattatcgc gcgcggtgtc atctatgtta ctagatcggg 1260 aagatccccg ggtaccgagctcgaatt 1287 9 1303 DNA Oryza sativa Pollen-specific gene 9 ccgggccatggcatccttta gaatggagga atttaagtga aattgagcta aactatgtga 60 acatcctatgaagttactgc attcaaggcg cccaacatga aatctattca ggttcccaag 120 ttgtgggcttccgtaacgtc aaaattcgac agatttctgg ctggctaaaa cacccacaac 180 ggcaataatagcctcgctcg tcaaaacatt cacccatttt tagcttggtc atcatcaaaa 240 gtaggatcaaatcaacaatc tgccttctct tcagccactc gatcccaacg gcatctccaa 300 cgattcctacttgaaggaca gccatggaaa tcctccaggt tccccaggtt acttatacca 360 cagctcgaatccgttccaaa ccaggccatt tcagtaccct cctctcacat tttccccaaa 420 taataatagaggaaggggaa aaacacattt gcagccacat catccatggc ctctctccgc 480 accattccggtgatcttcgg catcctcttc tatgtccttg ccagcactgc cactgccacc 540 gacgcaccagactacgtcgt ccaaggccgt gtctactgtg acacgtgccg cgccgagttc 600 gagaccaatgtcaccgagta tatcaagggt aaggaaattc ttttttgggt caggagtctg 660 caatgaaaatgctgaaatga ataacctccg atatatgagc agcagaactt aggaagacca 720 aagaactgcagagtttgtgc atcaatttgt aaacatgaaa cgctaacctg gttagaagtc 780 cagcattggctcacctgatc tcttgattgc aggtgccaag gtcaggctgg agtgcaagca 840 ctttggcaccgacaaggtcg agcgtgcgat tgacggtgtg actgatgaga ccgggacata 900 caagattgagctcaaggaca gccatgagga ggacatctgc gaggttgtcc tcgtccacag 960 cccccttgcaaactgctctg aaatcgaggc cgaaagggat cgtgcccgtg ttttgctcac 1020 caggaatgtcggcatctgtg acaacctgcg cttagccaac ccactcggct acctcaagga 1080 ctaccactgcccgtctgcgg cgctgctcaa gcagttcgac ctggctgatg atgataacga 1140 gtaatgcgatgatcgtcatg gaacctccgg agaggctgca ttaattataa atcagttaga 1200 ggcttgcaaaatagcatgga tctatctgaa aggcagaact aagcatatgt caaaacatga 1260 aatgtacactcatcactaag tactcacatg tgactacctg agg 1303 10 27 DNA Artificial SequenceDescription of Artificial Sequence 5′ PCR primer 10 acccatgtgagtttctttct tctccat 27 11 28 DNA Artificial Sequence Description ofArtificial Sequence 5′ PCR primer 11 ttataggaaa attccagcag ctcagcat 2812 26 DNA Artificial Sequence Description of Artificial Sequence 5′ PCRprimer 12 gaattccggg ccatggcatc ctttag 26 13 24 DNA Artificial SequenceDescription of Artificial Sequence 5′ PCR primer 13 ccatggatgatgtggctgca aatg 24 14 6741 DNA Artificial Sequence Description ofArtificial Sequence Cloning vector pPZP200 for plant transformation 14agtactttga tccaacccct ccgctgctat agtgcagtcg gcttctgacg ttcagtgcag 60ccgtcttctg aaaacgacat gtcgcacaag tcctaagtta cgcgacaggc tgccgccctg 120cccttttcct ggcgttttct tgtcgcgtgt tttagtcgca taaagtagaa tacttgcgac 180tagaaccgga gacattacgc catgaacaag agcgccgccg ctggcctgct gggctatgcc 240cgcgtcagca ccgacgacca ggacttgacc aaccaacggg ccgaactgca cgcggccggc 300tgcaccaagc tgttttccga gaagatcacc ggcaccaggc gcgaccgccc ggagctggcc 360aggatgcttg accacctacg ccctggcgac gttgtgacag tgaccaggct agaccgcctg 420gcccgcagca cccgcgacct actggacatt gccgagcgca tccaggaggc cggcgcgggc 480ctgcgtagcc tggcagagcc gtgggccgac accaccacgc cggccggccg catggtgttg 540accgtgttcg ccggcattgc cgagttcgag cgttccctaa tcatcgaccg cacccggagc 600gggcgcgagg ccgccaaggc ccgaggcgtg aagtttggcc cccgccctac cctcaccccg 660gcacagatcg cgcacgcccg cgagctgatc gaccaggaag gccgcaccgt gaaagaggcg 720gctgcactgc ttggcgtgca tcgctcgacc ctgtaccgcg cacttgagcg cagcgaggaa 780gtgacgccca ccgaggccag gcggcgcggt gccttccgtg aggacgcatt gaccgaggcc 840gacgccctgg cggccgccga gaatgaacgc caagaggaac aagcatgaaa ccgcaccagg 900acggccagga cgaaccgttt ttcattaccg aagagatcga ggcggagatg atcgcggccg 960ggtacgtgtt cgagccgccc gcgcacgtct caaccgtgcg gctgcatgaa atcctggccg 1020gtttgtctga tgccaagctg gcggcctggc cggccagctt ggccgctgaa gaaaccgagc 1080gccgccgtct aaaaaggtga tgtgtatttg agtaaaacag cttgcgtcat gcggtcgctg 1140cgtatatgat gcgatgagta aataaacaaa tacgcaaggg gaacgcatga aggttatcgc 1200tgtacttaac cagaaaggcg ggtcaggcaa gacgaccatc gcaacccatc tagcccgcgc 1260cctgcaactc gccggggccg atgttctgtt agtcgattcc gatccccagg gcagtgcccg 1320cgattgggcg gccgtgcggg aagatcaacc gctaaccgtt gtcggcatcg accgcccgac 1380gattgaccgc gacgtgaagg ccatcggccg gcgcgacttc gtagtgatcg acggagcgcc 1440ccaggcggcg gacttggctg tgtccgcgat caaggcagcc gacttcgtgc tgattccggt 1500gcagccaagc ccttacgaca tatgggccac cgccgacctg gtggagctgg ttaagcagcg 1560cattgaggtc acggatggaa ggctacaagc ggcctttgtc gtgtcgcggg cgatcaaagg 1620cacgcgcatc ggcggtgagg ttgccgaggc gctggccggg tacgagctgc ccattcttga 1680gtcccgtatc acgcagcgcg tgagctaccc aggcactgcc gccgccggca caaccgttct 1740tgaatcagaa cccgagggcg acgctgcccg cgaggtccag gcgctggccg ctgaaattaa 1800atcaaaactc atttgagtta atgaggtaaa gagaaaatga gcaaaagcac aaacacgcta 1860agtgccggcc gtccgagcgc acgcagcagc aaggctgcaa cgttggccag cctggcagac 1920acgccagcca tgaagcgggt caactttcag ttgccggcgg aggatcacac caagctgaag 1980atgtacgcgg tacgccaagg caagaccatt accgagctgc tatctgaata catcgcgcag 2040ctaccagagt aaatgagcaa atgaataaat gagtagatga attttagcgg ctaaaggagg 2100cggcatggaa aatcaagaac aaccaggcac cgacgccgtg gaatgcccca tgtgtggagg 2160aacgggcggt tggccaggcg taagcggctg ggttgtctgc cggccctgca atggcactgg 2220aacccccaag cccgaggaat cggcgtgacg gtcgcaaacc atccggcccg gtacaaatcg 2280gcgcggcgct gggtgatgac ctggtggaga agttgaaggc cgcgcaggcc gcccagcggc 2340aacgcatcga ggcagaagca cgccccggtg aatcgtggca agcggccgct gatcgaatcc 2400gcaaagaatc ccggcaaccg ccggcagccg gtgcgccgtc gattaggaag ccgcccaagg 2460gcgacgagca accagatttt ttcgttccga tgctctatga cgtgggcacc cgcgatagtc 2520gcagcatcat ggacgtggcc gttttccgtc tgtcgaagcg tgaccgacga gctggcgagg 2580tgatccgcta cgagcttcca gacgggcacg tagaggtttc cgcagggccg gccggcatgg 2640ccagtgtgtg ggattacgac ctggtactga tggcggtttc ccatctaacc gaatccatga 2700accgataccg ggaagggaag ggagacaagc ccggccgcgt gttccgtcca cacgttgcgg 2760acgtactcaa gttctgccgg cgagccgatg gcggaaagca gaaagacgac ctggtagaaa 2820cctgcattcg gttaaacacc acgcacgttg ccatgcagcg tacgaagaag gccaagaacg 2880gccgcctggt gacggtatcc gagggtgaag ccttgattag ccgctacaag atcgtaaaga 2940gcgaaaccgg gcggccggag tacatcgaga tcgagctagc tgattggatg taccgcgaga 3000tcacagaagg caagaacccg gacgtgctga cggttcaccc cgattacttt ttgatcgatc 3060ccggcatcgg ccgttttctc taccgcctgg cacgccgcgc cgcaggcaag gcagaagcca 3120gatggttgtt caagacgatc tacgaacgca gtggcagcgc cggagagttc aagaagttct 3180gtttcaccgt gcgcaagctg atcgggtcaa atgacctgcc ggagtacgat ttgaaggagg 3240aggcggggca ggctggcccg atcctagtca tgcgctaccg caacctgatc gagggcgaag 3300catccgccgg ttcctaatgt acggagcaga tgctagggca aattgcccta gcaggggaaa 3360aaggtcgaaa aggtctcttt cctgtggata gcacgtacat tgggaaccca aagccgtaca 3420ttgggaaccg gaacccgtac attgggaacc caaagccgta cattgggaac cggtcacaca 3480tgtaagtgac tgatataaaa gagaaaaaag gcgatttttc cgcctaaaac tctttaaaac 3540ttattaaaac tcttaaaacc cgcctggcct gtgcataact gtctggccag cgcacagccg 3600aagagctgca aaaagcgcct acccttcggt cgctgcgctc cctacgcccc gccgcttcgc 3660gtcggcctat cgcggccgct ggccgctcaa aaatggctgg cctacggcca ggcaatctac 3720cagggcgcgg acaagccgcg ccgtcgccac tcgaccgccg gcgcccacat caaggcaccc 3780tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg 3840gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg 3900ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat 3960actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg 4020aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc 4080tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 4140cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 4200gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 4260gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 4320gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 4380ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 4440atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 4500tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 4560ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 4620gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 4680ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 4740ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 4800agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 4860ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg catgatatat 4920ctcccaattt gtgtagggct tattatgcac gcttaaaaat aataaaagca gacttgacct 4980gatagtttgg ctgtgagcaa ttatgtgctt agtgcatcta atcgcttgag ttaacgccgg 5040cgaagcggcg tcggcttgaa cgaatttcta gctagacatt atttgccgac taccttggtg 5100atctcgcctt tcacgtagtg gacaaattct tccaactgat ctgcgcgcga ggccaagcga 5160tcttcttctt gtccaagata agcctgtcta gcttcaagta tgacgggctg atactgggcc 5220ggcaggcgct ccattgccca gtcggcagcg acatccttcg gcgcgatttt gccggttact 5280gcgctgtacc aaatgcggga caacgtaagc actacatttc gctcatcgcc agcccagtcg 5340ggcggcgagt tccatagcgt taaggtttca tttagcgcct caaatagatc ctgttcagga 5400accggatcaa agagttcctc cgccgctgga cctaccaagg caacgctatg ttctcttgct 5460tttgtcagca agatagccag atcaatgtcg atcgtggctg gctcgaagat acctgcaaga 5520atgtcattgc gctgccattc tccaaattgc agttcgcgct tagctggata acgccacgga 5580atgatgtcgt cgtgcacaac aatggtgact tctacagcgc ggagaatctc gctctctcca 5640ggggaagccg aagtttccaa aaggtcgttg atcaaagctc gccgcgttgt ttcatcaagc 5700cttacggtca ccgtaaccag caaatcaata tcactgtgtg gcttcaggcc gccatccact 5760gcggagccgt acaaatgtac ggccagcaac gtcggttcga gatggcgctc gatgacgcca 5820actacctctg atagttgagt cgatacttcg gcgatcaccg cttcccccat gatgtttaac 5880tttgttttag ggcgactgcc ctgctgcgta acatcgttgc tgctccataa catcaaacat 5940cgacccacgg cgtaacgcgc ttgctgcttg gatgcccgag gcatagactg taccccaaaa 6000aaacatgtca taacaagaag ccatgaaaac cgccactgcg ccgttaccac cgctgcgttc 6060ggtcaaggtt ctggaccagt tgcgtgacgg cagttacgct acttgcatta cagcttacga 6120accgaacgag gcttatgtcc actgggttcg tgcccgaatt gatcacaggc agcaacgctc 6180tgtcatcgtt acaatcaaca tgctaccctc cgcgagatca tccgtgtttc aaacccggca 6240gcttagttgc cgttcttccg aatagcatcg gtaacatgag caaagtctgc cgccttacaa 6300cggctctccc gctgacgccg tcccggactg atgggctgcc tgtatcgagt ggtgattttg 6360tgccgagctg ccggtcgggg agctgttggc tggctggtgg caggatatat tgtggtgtaa 6420acaaattgac gcttagacaa cttaataaca cattgcggac gtttttaatg tactgaatta 6480acgccgaatt gaattcgagc tcggtacccg gggatcctct agagtcgacc tgcaggcatg 6540caagcttagc ttgagcttgg atcagattgt cgtttcccgc cttcagttta aactatcagt 6600gtttgacagg atatattggc gggtaaacct aagagaaaag agcgtttatt agaataacgg 6660atatttaaaa gggcgtgaaa aggtttatcc gttcgtccat ttgtatgtgc atgccaacca 6720cagggttccc ctcgggatca a 6741 15 1246 DNA Artificial Sequence Descriptionof Artificial Sequence Construct- AT59BarnaseNos GST 15 ctgcaggggatttttttaat tacttgtatg ataattattt tcaatagacc tagagacttg 60 atatatactacgtttaataa tcatatgtag tatgtatgat taattaagta aatacaaaaa 120 tagttacctcaagttttaaa ggtgctattg ggtaattatc tcagtaaaaa taatattaga 180 tcaaggcaaaaataactgaa aatatccaga aaagaaggat taaacaaagg catccaaaat 240 ctataattgggttttttgga gaaatgacca tagagattta aatcaatggt tgtctaatct 300 atgttaattctcaatcctct attgactctt ctcatctcct tttctctctc cccagttcct 360 ggttattaaagcaatcaggt gattcaaatc tttaatcttt taatcccggc aggcctatct 420 gaaacaacaacctccgtttg aggttttgcc gggaaaatat aaagttcaca ggctttggtc 480 tctgcatttgcaatatattt accatggtac cggttatcaa cacgtttgac ggggttgcgg 540 attatcttcagacatatcat aagctacctg ataattacat tacaaaatca gaagcacaag 600 ccctcggctgggtggcatca aaagggaacc ttgcagacgt cgctccgggg aaaagcatcg 660 gcggagacatcttctcaaac agggaaggca aactcccggg caaaagcgga cgaacatggc 720 gtgaagcggatattaactat acatcaggct tcagaaattc agaccggatt ctttactcaa 780 gcgactggctgatttacaaa acaacggacc attatcagac ctttacaaaa atcagataac 840 gaaaaaaacggcttcctgcg gaggccgttt ttttcagctt tacataaagt gtgtaataaa 900 tttttcttcaaactctgatc ggtcaatttc actttccggn nnnctctaga ggatccgaag 960 cagatcgttcaaacatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg 1020 cgatgattatcatataattt ctgttgaatt acgttaagca tgtaataatt aacatgtaat 1080 gcatgacgttatttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat 1140 acgcgatagaaaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat 1200 ctatgttactagatcgggaa gatccccggg taccgagctc gaattc 1246 16 1213 DNA ArtificialSequence Description of Artificial Sequence Construct- OsPSPBarnaseNosGST 16 gaattccggg ccatggcatc ctttagaatg gaggaattta agtgaaattg agctaaacta60 tgtgaacatc ctatgaagtt actgcattca aggcgcccaa catgaaatct attcaggttc 120ccaagttgtg ggcttccgta acgtcaaaat tcgacagatt tctggctggc taaaacaccc 180acaacggcaa taatagcctc gctcgtcaaa acattcaccc atttttagct tggtcatcat 240caaaagtagg atcaaatcaa caatctgcct tctcttcagc cactcgatcc caacggcatc 300tccaacgatt cctacttgaa ggacagccat ggaaatcctc caggttcccc aggttactta 360taccacagct cgaatccgtt ccaaaccagg ccatttcagt accctcctct cacattttcc 420ccaaataata atagaggaag gggaaaaaca catttgcagc cacatcatcc atggtaccgg 480ttatcaacac gtttgacggg gttgcggatt atcttcagac atatcataag ctacctgata 540attacattac aaaatcagaa gcacaagccc tcggctgggt ggcatcaaaa gggaaccttg 600cagacgtcgc tccggggaaa agcatcggcg gagacatctt ctcaaacagg gaaggcaaac 660tcccgggcaa aagcggacga acatggcgtg aagcggatat taactataca tcaggcttca 720gaaattcaga ccggattctt tactcaagcg actggctgat ttacaaaaca acggaccatt 780atcagacctt tacaaaaatc agataacgaa aaaaacggct tcctgcggag gccgtttttt 840tcagctttac ataaagtgtg taataaattt ttcttcaaac tctgatcggt caatttcact 900ttccggnnnn ctctagagga tccgaagcag atcgttcaaa catttggcaa taaagtttct 960taagattgaa tcctgttgcc ggtcttgcga tgattatcat ataatttctg ttgaattacg 1020ttaagcatgt aataattaac atgtaatgca tgacgttatt tatgagatgg gtttttatga 1080ttagagtccc gcaattatac atttaatacg cgatagaaaa caaaatatag cgcgcaaact 1140aggataaatt atcgcgcgcg gtgtcatcta tgttactaga tcgggaagat ccccgggtac 1200cgagctcgaa ttc 1213

What is claimed is:
 1. A nucleic acid construct comprising a malegamete- or female gamete-specific promoter operably linked to a suicidegene, wherein said promoter and said suicide gene combination is linkedto a gene of interest.
 2. A nucleic acid construct comprising apollen-specific promoter or an ovule-specific promoter operably linkedto a suicide gene selected from the group consisting of barnase,tasselseed2 and diphtheria toxin A gene; wherein said promoter and saidsuicide gene combination is linked to a gene of interest selected fromthe group consisting of a gene coding for herbicide resistance,antibiotic resistance, insecticide resistance, nitrogen fixation,improved nutrition and cellulose content or other agronomic trait ofinterest.
 3. The nucleic acid construct of claim 1 wherein said promoteris selected from the group consisting of a pollen-specific promoter andan ovule-specific promoter.
 4. The nucleic acid construct of claim 1wherein said suicide gene is selected from the group consisting ofbarnase, tasselseed2 and diphtheria toxin A gene.
 5. The nucleic acidconstruct of claim 1 wherein said gene of interest is selected from thegroup consisting of a nucleic acid encoding herbicide resistance,antibiotic resistance, insecticide resistance, nitrogen fixation,improved nutrition and cellulose content.
 6. A vector comprising thenucleic acid construct of claim
 1. 7. A vector comprising the nucleicacid construct of claim
 2. 8. A host cell comprising the vector of claim6.
 9. A host cell comprising the vector of claim
 7. 10. A recombinantplant cell comprising the vector of any one of claims 6-7.
 11. Atransgenic plant comprising the vector of any one of claims 6-7.
 12. Thetransgenic plant of claim 11, wherein the transgenic plant ishemizygotic for the nucleic acid construct.
 13. A method for reducing oreliminating male transmission of a transgene locus in a plantcomprising: a) transforming a plant cell with a nucleic acid constructin which a male gamete-specific promoter is operably linked to a suicidegene, wherein said promoter and said suicide gene combination is linkedto a heterologous polynucleotide; b) propagating said transformed plantcell through meiosis to produce male gametes lacking said transgenelocus.
 14. A method for reducing or eliminating male transmission of atransgene locus in a plant comprising: a) transforming a plant cell witha nucleic acid construct in which a pollen-specific promoter is operablylinked to a suicide gene; i) wherein said suicide gene is selected fromthe group consisting of bamase, tasselseed2 and diphtheria toxin A gene;ii) wherein said promoter and said suicide gene combination is linked toa heterologous polynucleotide; iii) wherein said heterologouspolynucleotide is selected from the group consisting of DNA encodingherbicide resistance, antibiotic resistance, insecticide resistance,nitrogen fixation, improved nutrition and cellulose content; b)propagating said transformed plant cell through meiosis to produce malegametes lacking said transgene locus.
 15. A method for reducing oreliminating female transmission of a transgene locus in a plantcomprising: a) transforming a plant cell with a nucleic acid constructin which a female gamete-specific promoter is operably linked to asuicide gene, wherein said promoter and said suicide gene combination islinked to a heterologous polynucleotide; b) propagating said transformedplant cell through meiosis to produce female gametes lacking saidtransgene locus.
 16. A method for reducing or eliminating femaletransmission of a transgene locus in a plant comprising: a) transforminga plant cell with a nucleic acid construct in which an ovule-specificpromoter is operably linked to a suicide gene; i) wherein said suicidegene is selected from the group consisting of barnase, tasselseed2 anddiphtheria toxin A gene; ii) wherein said promoter and said suicide genecombination is linked to a heterologous polynucleotide; iii) whereinsaid heterologous polynucleotide is selected from the group consistingof DNA encoding herbicide resistance, antibiotic resistance, insecticideresistance, nitrogen fixation, improved nutrition and cellulose content.b) propagating said transformed plant cell through meiosis to producefemale gametes lacking said transgene locus.
 17. The transformed plantcell of claim 13, 14, 15 or 16, wherein the transformed plant cell ishemizygotic for the nucleic acid construct.
 18. A nucleic acid constructcomprising a male gamete- or female gamete-specific promoter operablylinked to a suicide gene wherein said promoter and said suicide genecombination is linked to a transposable element.
 19. The nucleic acidconstruct of claim 18, further comprising a transposase gene.
 20. Thenucleic acid construct of claim 18 or claim 19 further comprising a geneof interest.
 21. The nucleic acid construct of claim 20, wherein thegene of interest is associated with the transposable element.
 22. Anucleic acid construct in which a pollen-specific promoter or anovule-specific promoter is operably linked to a suicide gene selectedfrom the group consisting of bamase, tasselseed2 and diphtheria toxin Agene; wherein said promoter and said suicide gene combination is linkedto a transposon, wherein said transposon comprises a selectable markerselected from the group consisting of a gene coding for herbicideresistance, antibiotic resistance, insecticide resistance, nitrogenfixation, improved nutrition and cellulose content.
 23. The nucleic acidconstruct of claim 22 wherein said promoter is selected from the groupconsisting of a pollen-specific promoter and an ovule-specific promoter.24. The nucleic acid construct of claim 22 wherein said suicide gene isselected from the group consisting of barnase, tasselseed2 anddiphtheria toxin A gene.
 25. A vector comprising the nucleic acidconstruct of claim
 18. 26. A vector comprising the nucleic acidconstruct of claim
 22. 27. A host cell comprising the vector of claim18.
 28. A host cell comprising the vector of claim
 22. 29. A recombinantplant cell comprising the vector of claim 18 or claim
 22. 30. Therecombinant plant cell of claim. 29, wherein the recombinant plant cellis hemizygotic for the nucleic acid construct.
 31. A transgenic plantcomprising the vector of claim 18 or claim
 22. 32. The transgenic plantof claim 31, wherein the recombinant plant cell is hemizygotic for thenucleic acid construct.
 33. A method for enriching dispersedtransposition events in a population of plant cell progeny comprising:a) transforming a plant cell with the nucleic acid construct of any oneof claims 18-24 to produce a transformed plant cell; b) propagating saidtransformed plant cell through meiosis to produce plant cell progeny inwhich dispersed transposition events are enriched.
 34. The method ofclaim 33 further comprising: c) isolating said plant cell progeny inwhich dispersed transposition events are enriched.
 35. A plant cellisolated by the method of claim
 34. 36. A plant produced from the plantcell of claim
 35. 37. The plant cell of any one of claims 33, 34 or 35,wherein the plant cell is hemizygotic for the nucleic acid.
 38. Anucleic acid construct comprising a first promoter wherein the firstpromoter is a male gamete- or female gamete-specific promoter operablylinked to a suicide gene and further comprising a nucleic acid encodinga transposase and a nucleic acid encoding a transposon.
 39. The nucleicacid construct of claim 38, wherein the transposon comprises a secondpromoter operably linked to a selectable marker, wherein the selectablemarker is not a suicide gene.
 40. A nucleic acid construct in which apollen-specific promoter or an ovule-specific promoter is operablylinked to a suicide gene selected from the group consisting of bamase,tasselseed2 and diphtheria toxin A gene, wherein said promoter and saidsuicide gene combination is linked to a nucleic acid encodingtransposase; wherein said promoter and said suicide gene combinationlinked to said nucleic acid encoding transposase comprise a transgenelocus which further comprises a transposon; wherein said transposoncomprises a polynucleotide sequence encoding a member selected from thegroup consisting of herbicide resistance, antibiotic resistance,insecticide resistance, nitrogen fixation, improved nutrition andcellulose content.
 41. The nucleic acid construct of claim 40 whereinsaid promoter is selected from the group consisting of a pollen-specificpromoter and an ovule-specific promoter.
 42. The nucleic acid constructof claim 40 wherein said suicide gene is selected from the groupconsisting of bamase, tasselseed2 and diphtheria toxin A gene.
 43. Thenucleic acid construct of claim 40 wherein said transposon comprises apolynucleotide sequence encoding a member selected from the groupconsisting of herbicide resistance, antibiotic resistance, insecticideresistance, nitrogen fixation, improved nutrition and cellulose content.44. A vector comprising the nucleic acid construct of claim
 38. 45. Avector comprising the nucleic acid construct of claim
 40. 46. A hostcell comprising the vector of claim
 44. 47. A host cell comprising thevector of claim
 45. 48. The host cell of claim 46 or claim 47, whereinthe nucleic acid construct is hemizygotic.
 49. A recombinant plant cellcomprising the vector of any one of claims 44-45.
 50. The recombinantplant cell of claim 49, wherein the nucleic acid construct ishemizygotic.
 51. A transgenic plant comprising the vector of any one ofclaims 44-45.
 52. The transgenic plant of claim 51, wherein the nucleicacid is hemizygotic.
 53. A method for enriching stably dispersedtransposition events in a population of plant cell progeny comprising:a) transforming a plant cell with a nucleic acid construct of any one ofclaims 38-43 to produce a transformed plant cell; b) propagating saidtransformed plant cell through meiosis to produce plant cell progeny inwhich stably dispersed transposition events are enriched.
 54. The methodof claim 53 further comprising: c) isolating said plant cell progeny inwhich stably dispersed transposition events are enriched.
 55. A plantcell isolated by the method of claim
 54. 56. A plant produced from theplant cell of claim 55.